encapsulation of factor ix-engineered ......the work presented in this thesis was focused on design...

1

ENCAPSULATION OF FACTOR IX-ENGINEERED MESENCHYMAL STEM

CELLS IN ALGINATE-BASED MICROCAPSULES FOR ENHANCED VIABILITY

AND FUNCTIONALITY

ENCAPSULATION OF FACTOR IX-ENGINEERED MESENCHYMAL STEM

CELLS IN PROTEIN OR PEPTIDE MODIFIED ALGINATE MICROCAPSULES FOR

ENHANCED VIABILITY AND FUNCTIONALITY

By

BAHAREH SAYYAR, B.Sc. (Eng), M.A.Sc. (Eng)

A Thesis

Submitted to the School of Graduate Studies

in Partial Fulfilment of the Requirements

for the Degree

Doctor of Philosophy

McMaster University

© Copyright by Bahareh Sayyar, September 2012

ii

DOCTOR OF PHILOSOPHY (2012) McMaster University

(Biomedical Engineering) Hamilton, Ontario

TITLE: ENCAPSULATION OF FACTOR IX-ENGINEERED MESENCHYMAL

STEM CELLS IN PROTEIN OR PEPTIDE MODIFIED ALGINATE

MICROCAPSULES FOR ENHANCED VIABILITY AND FUNCTIONALITY

AUTHOR: Bahareh Sayyar

B.Sc. (Eng) (Sharif University of Technology)

M.A.Sc. (Eng) (University of Toronto)

SUPERVISOR: Dr. Gonzalo Hortelano

NUMBER OF PAGES: xiii, 144

iii

This thesis is dedicated to my husband, Masoud. I give my deepest

expression of love and appreciation for encouragements and support.

iv

ABSTRACT

The work presented in this thesis was focused on design and construction of novel

cell-loaded microcapsules by incorporation of bioactive molecules (proteins or peptides)

for potential application in hemophilia B treatment. The objective of this study was to

improve the viability and functionality of the encapsulated cells by creating biomimetic

microenvironments for cells that more closely mimic their physiological extracellular

matrix (ECM) environment.

Three cell-adhesive molecules were used in this work: fibrinogen and fibronectin,

two abundant proteins present in ECM, and arginine-glycine-aspartic acid (RGD) tri-

peptide, the minimal essential cell adhesion peptide sequence and the most widely studied

peptide for cell adhesion. Alginate, the most commonly used biomaterial used for cell

encapsulation, was combined with either of these molecules to create biomimetic

microcapsules. Non-modified alginate (control) and modified alginate matrices were used

to encapsulate the factor IX (FIX) secreting cells for protein delivery. In this work, FIX-

engineered cord blood-derived human mesenchymal stem cells CB MSCs were used as a

cell source for FIX delivery.

Our data suggested that fibrinogen-alginate, fibronectin-alginate and RGD-

alginate microcapsules improved the viability of encapsulated MSC and are applicable in

cell therapy technologies. However, fibrinogen-alginate and fibronectin-alginate

microcapsules more significantly enhanced the proliferation and protein secretion from

the encapsulated cells and may have potential for FIX delivery for hemophilia B and

other inherited or acquired protein deficiencies. RGD-alginate microcapsules can

v

potentially be used for other tissue engineering applications with the aim of enhanced

viability and attachment of the enclosed cells. Differentiation studies showed the

osteogenic (but not chondrogenic or adipogenic) differentiation capability of FIX-

engineered CB MSCs and their efficient FIX secretion while encapsulated in fibrinogen-

alginate and fibronectin-alginate microcapsules.

vi

ACKNOWLEDGEMENTS

I have many people to acknowledge with the completion of this thesis. I would

like to take this opportunity to express my sincere appreciation to all who have helped

and supported me.

First to my supervisor, Dr. Gonzalo Hortelano, I am immensely grateful to you for

always being there with great guidance and encouragement. You have continually

supported me through my journey to complete this work, while giving me the freedom to

pursue my research interests. Having you, a great researcher and an outstanding person,

as my supervisor enabled me not only to learn about the field but also how to face

problems in general. You have shaped the researcher as I am and I am extremely grateful

to you. My sincere gratitude is also extended to my supervisory committee members Dr.

Murray Potter and Dr. Harald Stöver. Thank you for your invaluable advice and

suggestions. It has been a privilege to have you as my academic committee members. I

could not have asked for a better experience and feel so fortunate for the opportunity to

learn from such accomplished researchers.

I would like to continue by acknowledging the researchers at Hortelano lab. First,

I would like to thank Jianping Wen. I am extremely grateful to you. You are not only a

great researcher but also a wonderful friend who has always supported me and never

hesitated to provide advice and ideas. Thanks to Megan Dodd, for being such a great

friend and colleague. I am lucky to share my Ph.D. studies experience with you. Thanks

to the other members of Hortelano lab Azra Markar and Bobby Dhadwar, it was great

working with you.

vii

Thanks to my friends and colleagues at the School of Biomedical Engineering for

a memorable graduate experience. Especially to Sara Alibeik for always being there for

me and supporting me. I have had a chance of working with and learning form

researchers and faculty members from Chemical Engineering, Mechanical Engineering

and Chemistry Departments. Dr. Todd Hoare, Siawash Shinwary, Leah R. Kesselman,

Shirley Ma, and Casey Gardner, I enjoyed working with you and value your help.

I would also like to gratefully acknowledge the support of all funding agencies

that support this project: Canadian Blood Services (CBS), Natural Resources and

Engineering Research Council of Canada (NSERC-CREATE/IDEM), Ontario Graduate

Scholarships for OGS and OGSST scholarships, McMaster University for the research

scholarship, International Excellence Award, Yates scholarship and GSA scholarship, and

Canadian Biomaterials Society for several travel awards.

Thank you to all my family and friends. Especially thanks to my dear parents

Masoud and Azar for raising me to be who I am. You taught me the value of hard work

and excellent education. To my lovely sister, Parastoo. You are my closest friend and I

am extremely lucky to have you in my life.

Last but certainly not least, to Masoud Golshan, my husband, my best friend, my

life. You are the best thing that ever happened to me and I cannot even start to thank you

for who you are and what you have done. There are no words to convey how much I

appreciate your encouragements, love, and support. I would like to dedicate this thesis to

you.

viii

TABLE OF CONTENTS

ABSTRACT ....................................................................................................................... iv

ACKNOWLEDGEMENTS ............................................................................................. vi

TABLE OF CONTENTS .............................................................................................. viii

LIST OF FIGURES ........................................................................................................... x

LIST OF ABBREVIATIONS .......................................................................................... xi

CHAPTER 1: INTRODUCTION ..................................................................................... 1

1.1 OVERVIEW ................................................................................................................. 1

1.2 LITERATURE REVIEW ........................................................................................... 3

1.2.1 Hemophilia ............................................................................................................. 3

1.2.1.1 Current Treatment ............................................................................................ 5

1.2.1.2 Gene Therapy for Hemophilia ......................................................................... 6

1.2.2 Cell Therapy Strategies for Hemophilia Treatment .......................................... 8

1.2.2.1 Cell Encapsulation ......................................................................................... 10

1.2.2.2 Cell Source ..................................................................................................... 13

1.2.2.2.1 Mesenchymal Stem cells ......................................................................... 14

1.2.2.3 Biomaterials for Cell Encapsulation .............................................................. 16

1.2.2.3.1 Alginate ................................................................................................... 17

1.2.2.3.2 Other Polymers and Biomaterials ........................................................... 19

1.2.2.4 Biomaterials Modification for Bioactivity ..................................................... 21

1.2.2.4.1 Cell-Matrix Interactions .......................................................................... 22

1.2.2.4.2 Modification with Cell-Adhesive Molecules .......................................... 25

1.3 References ................................................................................................................... 31

CHAPTER 2: OBJECTIVES AND CONTRIBUTIONS TO ARTICLES ................ 41

2.1 OBJECTIVES ........................................................................................................ 41

2.2 CONTRIBUTIONS TO ARTICLES ................................................................... 43

ix

CHAPTER 3: ENCAPSULATION OF FIX-ENGINEERED HUMAN

MESENCHYMAL STEM CELLS IN FIBRINOGEN-ALGINATE

MICROCAPSULES ENHANCES CELL VIABILITY AND FIX SECRETION ..... 44


MESENCHYMAL STEM CELLS IN RGD-ALGINATE VS. FIBRINOGEN-

ALGINATE MICROCAPSULES FOR ENHANCED FIX SECRETION ................ 75

CHAPTER 5: FIBRONECTIN-ALGINATE MICROCAPSULES IMPROVE CELL

VIABILITY AND PROTEIN SECRETION OF ENCAPSULATED FIX-

ENGINEERED HUMAN MESENCHYMAL STEM CELLS .................................. 102

CHAPTER 6: SUMMARY AND RECOMMENDATIONS FOR FUTURE WORK

.......................................................................................................................................... 132

6.1 Summary ............................................................................................................... 132

6.2 Recommendations for Future Work .................................................................. 135

6.3 references: ............................................................................................................. 136

APPENDIX A : Experimental Methods ...................................................................... 137

A.1 Cell encapsulation procedure ................................................................................. 137

A.2 LIVE/DEAD viability kit for encapsulated cells .................................................. 138

A.3 Statistical analysis ................................................................................................... 142

APPENDIX B : Publications and Awards ................................................................... 143

x

LIST OF FIGURES

Figure 1.1 Scheme of blood coagulation cascade. E-extrinsic pathway; I-intrinsic

pathway; FC-final common pathway; VKD-vitamin K dependent; SP-serine protease;

TG-transglutaminase; NEC-non-enzymatic pathway. Reprinted from (Orlova et al. 2012)

Figure 1.2 In vivo gene therapy. Reprinted from:

http://genetherapy.yolasite.com/process.php/15/08/12

Figure 1.3 Cell microencapsulation. A, Nutrients, oxygen, stimuli and therapeutic agents

diffuse from the membrane while antibodies and immune cells are excluded (Orive et al.

2003). B, Cord blood-derived mesenchymal stem cells encapsulated in alginate-PLL-

alginate microcapsules.

Figure 1.4 Alginate polysaccharide that consists of two guluronic acid and two

mannuronic acid residues with (1,4)-linkages (Augst et al. 2006a).

Figure 1.5 Immobilized ligands lead to cell adhesion and survival (Hersel et al. 2003).

Figure 1.6 The RGD sequence and its molecular formula (Hersel et al. 2003).

Figure 1.7 Peptide coupling to alginate backbone (J A Rowley et al. 1999). Amide bond

is formed by the cabodiimide through the carboxyl functional group of alginate and N-

terminal amide of the RGD peptide.

Figure A-1 Cell encapsulation procedure

http://genetherapy.yolasite.com/process.php/15/08/12

xi

LIST OF ABBREVIATIONS

APA: alginate-poly-L-lysine-alginate

ATTP: activated partial thromboplastin time

BMP: bone morphogenetic protein

CDK: cyclin-dependent kinase

CNS: central nervous system

E: extrinsic pathway (coagulation cascade)

ECM: extracellular matrix

EDC: 1-ethyl-(dimethylaminopropyl) carbodiimide

ESCs: embryonic stem cells

FAK: focal adhesion kinase

FC: final common pathway (coagulation cascade)

FVIII: factor VIII

FIX: factor IX

FX: factor X

G: guluronic acid

HA: hyaluronic acid

HEMA: (hydroxyethyl)methacrylate

I: intrinsic pathway (coagulation cascade)

M: mannuronic acid

MAPK: mitogen activated protein kinase

xii

MSC: mesenchymal stem cell

NEC: non-enzymatic pathway

OPD: o-Phenylenediamine

PBS: phosphate buffer saline

PEG: poly(ethylene glycol)

PI3K: phosphoinositide 3-kinase

RGD: arginine-glycine-aspartic acid

sulfo-NHS; N-hydroxysulfosuccinimide

VKD: vitamin K dependent

Ph.D. Thesis-B. Sayyar McMaster University-Biomedical Engineering

1

CHAPTER 1: INTRODUCTION

1.1 OVERVIEW

The work presented in this thesis is aimed at design and construction of novel cell-

loaded microcapsules for enhanced viability and functionality of the enclosed FIX

secreting cells for a potential application in hemophilia B treatment. This requires an

understanding of hemophilia B disease and its current treatment and limitations, cell

encapsulation as a cell therapy strategy and its appropriate choice of cells and

biomaterials, knowledge of cell-biomaterial interactions and the methods of biomaterials

modification to accommodate these interactions. This chapter will provide a review of all

these areas and will discuss the cell-loaded microcapsules that have been used for FIX

delivery.

Hemophilia B is an X-linked monogenic disorder caused by deficiencies in

coagulation factor IX (FIX) with a prevalence of 1:30,000 males (Bowen 2002).

Hemorrhagic episodes can be prevented or treated by injection of plasma-derived or

recombinant FIX, which is limited due to high cost, and unpredictable shortages of the

therapeutic proteins. Being a monogenic disorder, hemophilia B is an excellent candidate

for gene therapy. Moreover, there is a relatively low threshold for success. Modest

elevation of FIX levels is sufficient to improve the clinical symptoms significantly.

Current gene therapy protocols are often based on direct injection of viral or non-viral

vectors for FIX delivery that may be associated with safety concerns. Cell therapy

strategies offer several advantages including elimination of the need to modify the


2

patient’s genome, ex vivo screening of the cells before implantation, and sustained release

of therapeutic agents from the implanted cells.

Cell microencapsulation technology continues to hold significant promise for cell

therapy strategies. The continuous delivery of therapeutic proteins to the host by the

immunoisolated cells is potentially a convenient and cost-effective strategy to treat a

variety of protein deficiency disorders including hemophilia B. Nevertheless, this

technology has not completely lived up to expectations mainly due to the compromised

viability and functionality of the enclosed cells.

Ex vivo gene therapy protocols were often based on autologous cell implantations

to prevent cell rejection (Grossman et al. 1994). However, the need to genetically modify

each patient’s cell is labor-intensive and costly. Instead of engineering autologous cells to

secrete FIX, we envisioned creating universal FIX-secreting cell lines that could be

implanted in hemophilic patients by enclosing these cells in immunoisolation host

compatible microcapsules. Engineered mesenchymal stem cells (MSCs) are attractive

target cell populations for hemophilia gene therapy because they are readily accessible for

ex vivo genetic modification and are capable of differentiating into multiple lineages of

the mesenchyme. Moreover, it has been observed that MSCs are immunoprivileged and,

more importantly, display immunomodulatory capabilities (A. Goren et al. 2010).

Although alginate is the most widely employed material for cell encapsulation,

success has been limited due largely to the fact that it does not provide the enclosed cells

with attachment sites and does not mimic the physiological location of the normal cell

milieu.


3

We hypothesized that designing biomimetic microcapsules by incorporation of

cell-adhesive molecules, either peptides or proteins, might promote the viability and

functionality of the enclosed FIX-engineered MSCs and enhance the FIX secretion

profile. Fibrinogen-alginate, RGD-alginate, and fibronectin-alginate microcapsules are

constructed in this study to evaluate their effect on viability and functionality of the

enclosed cells. This study is predicted to represent an encouraging advance toward long-

term cure of this progressively debilitating, life-threatening bleeding disorder.

1.2 LITERATURE REVIEW

1.2.1 Hemophilia

Hemophilia is an X-linked inherited disorder that is characterized by impaired

blood coagulation as a result of deficiency or destruction of proteins that are involved in

the blood coagulation pathway (Bolton-Maggs & K. J. Pasi 2003; Bowen 2002).

Deficiencies in coagulation factors VIII and IX, (FVIII) and (FIX), are the two most

common forms of hemophilia known as hemophilia A and B, respectively. Hemophilia A

is the most common form of this disease affecting approximately 1:5,000 males. The

incidence of hemophilia B is 1:30,000 males (Bowen 2002). The frequent bleeding

episodes experienced by hemophilia B patients may be of immediate clinical importance

and are also associated with a range of long-term clinical disorders including

musculoskeletal problems (Gater et al. 2011).


4

Figure 1.1 Scheme of blood coagulation cascade. E-extrinsic pathway; I-intrinsic pathway; FC-final

common pathway; VKD-vitamin K dependent; SP-serine protease; TG-transglutaminase; NEC-non-

enzymatic pathway.

Reprinted from (Orlova et al. 2012)

FIX is a zymogen of serine protease produced in the liver. The inactive precursor

protein is processed in endoplasmic reticulum and Golgi where it is modified and secreted

into the blood stream. Circulated FIX, 57 kDa, takes part in the coagulation cascade in

presence of Ca+2

after specific proteolytic cleavage by the activated factor XI (FXIa; of

the contact pathway) or the activated factor VII (FVII; of the tissue factor pathway). FIXa

then hydrolyses one arginine-isoleucine bond on factor X to form the activated factor X

(FXa). Catalytic efficiency of FIXa is greatly increased by the cofactor FVIIIa (Orlova et

al. 2012). The non-covalent complex of FIXa, FVIIIa, and FX is a major signal

amplification loop in coagulation pathway (Figure 1.1)


5

The impact of hemophilia is determined by the severity of the condition. Patients

with mild condition (0.05-0.4 IU/ml of clotting factors) experience abnormal bleeding in

response to surgeries. However, patients with moderate hemophilia (0.01-0.05 IU/ml)

experience bleeding in response to relatively minor trauma and severe hemophiliacs

(<0.01 IU/ml) experience frequent spontaneous bleeds (White et al. 2001). At the same

time, classification based on clinical symptoms has been used since occasionally, patients

with FVIII or FIX levels<1% appear to be clinically moderate or mild as they do not

exhibit spontaneous bleeding and conversely, occasional patients with FVIII or FIX levels

of 1-5% may be clinically severe and have frequent spontaneous bleedings (White et al.

2001).

1.2.1.1 Current Treatment

Traditionally, hemophilia has been associated with significant mortality.

However, advances in hemophilia therapeutics over the past two decades can now

manage the condition. The aim of hemophilia treatments is to prevent or control bleeding

episodes by replacing the missing or defective coagulation factors. The modern treatment

of hemophilia started in 1970s and currently includes injecting variety of plasma-derived

or recombinant protein products (Coppola et al. 2012). Only 20% of the world’s

population can afford the treatment and therefore, hemophilia B remains lethal in poor

countries (Evatt et al. 2004). Considering the risk of transmission of blood-borne

diseases, recombinant FIX has been widely used in some countries but at a significantly

higher cost than plasma-derived FIX (White et al. 2001). Although the current treatments

considerably improve patients’ quality of life and prolong the life expectancy, they have


6

their own limitations. Beside the high cost associated with the current treatments, since

the treatment is non-curative, patients are always at the risk of bleeding episodes and

chronic joint damage. Also, the short half-life of the clotting factors demands repeated

infusion of large dosed of these proteins. Consequently, some patients develop antibodies

to supplemented FVIII or FIX that can render the further therapy extremely costly or non-

effective (Marinee K Chuah et al. 2012).

1.2.1.2 Gene Therapy for Hemophilia

Beginning 1980s the strategy of disease treatment by transferring the therapeutic

DNA began to be investigated. It was soon noted that hemophilia could be an ideal

condition to be treated by what came to be named gene therapy (E. Tuddenham 2012).

Currently, gene therapy offers an attractive alternative to the current costly and life-long

treatment of this disease (Marinee K Chuah et al. 2012; E. Tuddenham 2012; Pierce et al.

2007; K A High 2001; K A High 2011; Mátrai et al. 2010; T Vandendriessche & M K

Chuah 2012). The ultimate goal of gene therapy techniques is to replace the defective

gene with a corrected version for lifetime elimination of a disease (S. L. Murphy &

Katherine A High 2008; E. Tuddenham 2012) (Figure 1.2).


7

Figure 1.2 In vivo gene therapy.

Reprinted from: http://genetherapy.yolasite.com/process.php/15/08/12

Being a monogenic disorder, hemophilia B is an excellent candidate for gene

therapy. Functional part of the gene is small enough to fit into the modified vectors.

Moreover, there is a relatively low threshold for success. Modest elevation of FIX levels

to a few percent of normal is sufficient to improve the clinical symptoms significantly

(Manno et al. 2003). Finally, there are several validated small and large animal

hemophilic models for preclinical analysis (Marinee K Chuah et al. 2012). There have

been several gene therapy studies for hemophilia B. Currently, successful human gene

therapy methods involve direct injection of viral vectors for FIX delivery (Nathwani et al.

2011). Many viral and non-viral gene delivery systems with the ability to affect a variety

of mammalian cells have been modified to be used as a transfer agent for therapeutic

DNA and each have their own strengths and weaknesses (Marinee K Chuah et al. 2012;


8

E. Tuddenham 2012). Based on level and duration of expression and immune response

considerations, adeno associated viruses (AAV) and lentiviral vectors are among the most

promising vectors to date for hemophilia gene therapy (Marinee K Chuah et al. 2012).

Hepatocytes, muscle cells and hematopoietic stem cells (HSC) are the most attractive

target cells for in vivo trials in regards to their ability to secrete the clotting factors

(Marinee K Chuah et al. 2012; Mingozzi et al. 2003; K A High 2011; Mátrai et al. 2010).

The most recent human clinical trial in this regard demonstrates that in vivo

peripheral-vein injection of FIX-AAV8 leads to long-term expression of the FIX

transgene at therapeutic levels (Nathwani et al. 2011). Convincing results from the current

clinical trials confirm that the gene therapy strategies are effective for hemophilia B

patients. However, there are several associated safety concerns with in vivo injection of

viral vectors such as immune mediated AAV-capsid-induced elevations in

aminotransferase levels, immune mediated clearance of AAV-transduced hepatocytes or

transient hepatic disfunction (Nathwani et al. 2011; E. Tuddenham 2012; Marinee K

Chuah et al. 2012; Check 2003).

For Hemophilia B, safety of the novel more effective treatments is of great

importance.

1.2.2 Cell Therapy Strategies for Hemophilia Treatment

Cell therapy strategies offer several advantages to in vivo gene therapy. Cells can

act as drop depots enabling the delivery of therapeutic agents over an extended period of

time (Dove 2002). Cells are able to secrete and deliver therapeutic agents in response to


9

external stimuli, which is of great importance in maintenance of homeostasis in patients

with chronic diseases such as diabetes (Lim & Sun 1980). Moreover, they can secrete

variety of proteins and cytokines (Browne & Al-Rubeai 2007).

Previous ex vivo gene therapy protocols were often based on autologous

recombinant cell to prevent cell rejection (Grossman et al. 1994). However, the need to

genetically modify each patient’s cell is labor-intensive and costly. Therefore, it is of

great interest to explore the use of genetically engineered allogeneic cells for their

potential use.

It has been previously demonstrated that foreign cells can act as self-sustained

source of functional clotting factors (Bontempo et al. 1987; Gordon et al. 1998; Miao

2012; Kumaran et al. 2005; Madeira et al. 2009). Some cells have natural ability for FVIII

or FIX production and some can be genetically engineered to secrete high levels of either

proteins. Therefore, with only a small number of cells needed to achieve a therapeutic

protein secretion, feasible number of cells can be delivered for sustained and therapeutic

release. As a safety advantage, cell therapy strategies do not modify the genome of the

patients and the ex vivo cell screening enables the inspection of cells prior to

implantation.

The cell implantation strategy is as important for cell-based therapy as the ability

of cells to secrete the therapeutic agents. Cells directly injected into the patients

experience immune rejection and rapid decrease in cell viability (Dove 2002). Immune

suppressants may reduce the risk of immune rejection but the patient becomes more

vulnerable to other infections (Glynne et al. 2000). Also, the transplanted cells are not


10

well protected from mechanical stress or are not surrounded by the proper

microenvironment which may result in the rapid loss of viability and functionality

(Nagata et al. 2001). Instead of directly implanting the recombinant cells into the patients,

biomaterials can be used to enclose the cells within a physical barrier for better

mechanical and biological protection.

1.2.2.1 Cell Encapsulation

Cell encapsulation technology is based on immobilization of cells within a semi-

permeable membrane which protects the encapsulated cells from mechanical stress and

against antibodies and cytotoxic cells of the host immune system while allowing secretion

of therapeutic agents (Orive et al. 2003; Orive et al. 2004).

Figure 1.3 Cell microencapsulation. A, Nutrients, oxygen, stimuli and therapeutic agents diffuse from the

membrane while antibodies and immune cells are excluded (Orive et al. 2003). B, Cord blood-derived

mesenchymal stem cells encapsulated in alginate-PLL-alginate microcapsules.


11

In 1964, T.M.S. Chang proposed using an ultra thin polymeric membrane to

encapsulate transplanted cells for immune protection and introduced the term “artificial

cells” to define this concept (CHANG 1964). Since then, there has been considerable

progress toward understanding the biological and chemical requirements for successful

cell encapsulation. Implantation of cells enclosed in biocompatible, semi-permeable

microcapsules leads to continuous delivery of therapeutic proteins and to protect cells

from hosts’ immune response (Lim & Sun 1980; Koo & T. M. Chang 1993; P L Chang et

al. 1993; Cieslinski & David Humes 1994; Wong & T. M. Chang 1986; Aebischer et al.

1994; Aebischer et al. 1986; Orive et al. 2006; Orive et al. 2009; Thakur et al. 2010; G

Hortelano et al. 2001; G Hortelano et al. 1996; G Hortelano et al. 1999; Wen et al. 2006;

Wen et al. 2007). In the last few years, the applicability of this technology has been

shown for treatment of variety of endocrine diseases such as diabetes mellitus (Lim &

Sun 1980), hemophilia B (Wen et al. 2006; Wen et al. 2007), anemia (Koo & T. M.

Chang 1993), dwarfism (P L Chang et al. 1993), kidney (Cieslinski & David Humes

1994) and liver failure (Wong & T. M. Chang 1986), and central nervous system

(Aebischer et al. 1994) and pituitary insufficiencies (Aebischer et al. 1986). More

recently, microencapsules have been used as biodegradable scaffolds for stem cell

proliferation and differentiation as well as in vivo administration (Santos et al. 2009). As

shown in previous studies, application of cell encapsulation for hemophilia treatment

potentially results in reduction or even elimination of chronic administration of

immunosuppressants and therefore the serious risks associated with these drugs can be

avoided. Also, they allow a sustained and controlled delivery of therapeutic products. The


12

host genome is not modified and most of the microcapsules can be removed should it

become necessary to reverse the treatment. As an additional safety measure, recombinant

cells may be thoroughly characterized for unwanted genetic rearrangements before

encapsulation. To overcome the challenge of engineering cells from each individual, a

universal cell line can be engineered and encapsulated in biocompatible microcapsules to

be implanted in different patients. Cell encapsulation is one of the most important

methods for cell-based therapy and continuous drug delivery. Yet, despite some

promising results in animal studies, the field has not lived up to complete expectations.

Limitations of application of cell encapsulation of cell-based therapies include the

immunogenicity of the encapsulated cells, the selection of clinical grade biomaterials for

microcapsule fabrication, and the development of reproducible assays to measure the

encapsulated cell viability and protein secretion, and microcapsules biocompatibility

(Santos et al. 2009).

Encapsulated non-autologous cells secrete cytokines which evoke host immune

response and lead to inflammation surrounding the microcapsules which leads to

decreased viability of the encapsulated cells (G Hortelano et al. 1999). One solution to

reduce the host immune response is administration of anti-inflammatory drugs along with

implantation (G Hortelano & P L Chang 2000; M. Liu & Han 2008). The other approach

is to replace the cell lines commonly used for cell encapsulation with naïve cells such as

stem cells.


13

1.2.2.2 Cell Source

Various types of allogenic cells have been used in hemophilic cell-based

therapies. It was previously shown that active human FIX can be delivered from alginate-

based microcapsules containing human FIX secreting Ltk- human fibroblasts in vitro (H.

W. Liu et al. 1993). However, since the fibroblasts continued to proliferate and filled the

microcapsule, viability of the enclosed cells decreased significantly with time. Limited

exchange of nutrients and metabolic waste at the core of microcapsules led to necrosis

and low cell viability. In later studies, FIX-engineered myoblasts were used as a cell

source for encapsulation (G Hortelano et al. 1996; G Hortelano et al. 1999; G Hortelano

et al. 2001). Myoblasts can be terminally differentiated to myotubes and the encapsulated

non-proliferative myoblasts are known as stable cells for FIX delivery. Nude hemophilic

mice were implanted with microcapsules enclosing recombinant myoblasts secreting

human FIX. The activated partial thromboplastin time (APTT) was shortened

significantly in treated hemophilia mice and was coincided with appearance of human

FIX in mice plasma. On the other hand, plasma of immunocompetent hemophilic mice

similarly treated with microcapsules demonstrated a transient shortening of the APTT,

which was coincided with transient presence of human FIX in plasma. It was shown that

86% of nude mice implanted with microcapsules developed tumors with myoblast origin

(G Hortelano et al. 2001). Therefore, although these studies demonstrate the feasibility of

application of recombinant cell encapsulation for hemophilia B treatment, they highlight

the importance of choosing safe cell lines to prevent tumor development and also

decrease the immune response.


14

Transplanted fetal cells usually do not trigger immune response in a host and thus

can be used as a suitable cell source for ex vivo gene therapy applications (Dekel et al.

1999; Henningson et al. 2003). Wen et al. examined the implantation of microcapsules

enclosing recombinant G8 murine myoblasts of fetal origin secreting human FIX (Wen et

al. 2006). Encapsulated murine myoblasts displayed a non-antigenic behavior and

delivered therapeutic human FIX in immunocompetent mice for up to 120 days. Since the

ultimate goal was to develop a treatment for hemophilic patients, in a later study, they

evaluated the encapsulation of recombinant human primary myoblasts to deliver human

FIX in mice (Wen et al. 2007). Human FIX delivery was transient and became

undetectable on day 14. Concurrently, anti-human FIX antibodies were detected. The

APTT results showed that implantation of encapsulated recombinant human primary

myoblasts can partially correct the symptoms of murine hemophilia B. However, viability

of cells retrieved on day 60 was below 5%. This highlights the need to improve the long-

term viability of the encapsulated cells as a way to enhance the efficiency of this

treatment.

1.2.2.2.1 Mesenchymal Stem cells

Mesenchymal stem cells (MSCs) are a kind of adult stem cells that have gained a

great amount of interest during the last decade in regenerative medicine applications.

MSCs were first discovered by Friedenstein et al. in 1968 and were characterized as an

adherent fibroblast-like cell population in bone marrow capable of differentiating into

bone (Friedenstein et al. 1968). Since then, MSCs have been isolated from various tissues


15

such as adipose tissue, peripheral blood, umbilical cord blood, and amniotic fluid (S.

Wang et al. 2011). The International Society of Cellular Therapy defined MSCs in 2006

as follows: (1) they must be adherent to plastic under tissue culture conditions; (2) must

express certain cell surface markers such as CD73, CD90, and CD105 and lack the

expression of markers CD45, CD34, CD14, or CD11b, CD 79a or CD19 and HLA-DR;

(3) have the capacity to differentiate into osteoblasts, chondrocytes, and adipocytes under

in vitro conditions (Dominici et al. 2006). MSCs exert their therapeutic potentials through

some key potentials as follows: their capacity to differentiate into osteoblasts,

chondrocytes, adipocytes, hepatocytes, cardiomyocytes, neural and endothelial cells; their

ability to secrete bioactive molecules that inhibit inflammation and stimulate recovery of

injured cells; their lack of immunogenicity and their ability to perform

immunomodulatory functions (S. Wang et al. 2011).

The most interesting feature of MSCs is their hypoimmunogenic potential. MSCs

are characterized by low levels of major histocompatibility complex type I (MHC-I)

surface antigens and the lack of MHC-II antigens, FasL and B7-1, B7-2, CD40 or CD40L

(Gebler et al. 2011; Barry et al. 2005). Also, MSCs express the Toll-like receptors (TLRs)

which affect their immunomudulatory properties (Gebler et al. 2011). Therefore, because

of their low immunogenicity, MSCs are poorly recognized by human leukocyte antigen

(HLA)-incompatible hosts (Uccelli et al. 2006). Additionally, MSCs are known to

suppress cytotoxic or helper T cells (Uccelli et al. 2006; Beyth et al. 2005;

Klyushnenkova et al. 2005; Nauta & Fibbe 2007). Therefore, using the MSCs as an


16

alternative cell line for cell encapsulation may resolve the drawback associated with the

non-autologous cell implantation that is evoking inflammation around the microcapsules.

1.2.2.3 Biomaterials for Cell Encapsulation

By improvement of biomedicine, millions of patients have benefited from innovative

biomaterial-based products which have permitted early diagnosis, less invasive and quick

procedures, and fewer stays in hospitals (Santos et al. 2009). In this context, cell

encapsulation has gained a great interest in the field of cell therapy for tissue engineering

or drug delivery applications. One of the main issues in designing the microencapsulation

device is the choice of an appropriate material in terms of its stability and

biocompatibility (Santos et al. 2009). Moreover, preserving the viability and functionality

of the enclosed cells is of great importance if a therapeutic aim is anticipated.

Hydrogels are hydrophilic polymeric networks that may absorb in water from 10-

20% up to thousand times their dry weight (Hoffman 2002). Hydrogels posses several

properties that make them appealing for application in cell encapsulation (Schmidt et al.

2008). They are similar to extracellular matrices in terms of physical structure and

morphology (Weber et al. 2007). Additionally, due to their hydrophilic properties, they

represent the most biocompatible features compared to other biomaterials. Both naturally

derived and synthetic hydrogels have been used in cell encapsulation including alginate,

agarose, hyaluronic acid, chitosan, poly(hydroxyethyl)methacrylate (HEMA),

poly(ethylene glycol) (PEG) (Lim & Sun 1980; Khademhosseini et al. 2006; Karoubi et

al. 2009; Weber et al. 2006; Mokrý et al. 2000). While synthetic hydrogels present a more


17

reproducible and consistent composition, natural occurring hydrogels usually demonstrate

a better biocompatibility (Angelova & D Hunkeler 1999). Biocompatibility has been

defined as the ability of a biomaterial to perform with an appropriate host response in a

specific application (Santos et al. 2009). For the case of cell microencapsulation, the

interaction of the biomaterial with the enclosed cells should also be considered and both

aspects need to be considered if the long-term survival and functionality of the enclosed

cells are intended.

1.2.2.3.1 Alginate

Alginate is by far the most commonly used biomaterial in cell microencapsulation

(Santos et al. 2009). This is due to its capacity to form excellent gels in mild conditions as

well as its great in vivo biocompatibility. The encapsulation procedure can be performed

at room temperature, at physiological pH and using isotonic solutions, which results in

easier manageability of the whole process and higher viability of cells during capsule

elaboration.

Alginate is a polysaccharide isolated from brown algae such as Laminaria

hyperborean and lessonia. It is a linear unbranched copolymer that contains

homopolymeric blocks of (1,4)-linked -D-mannuronic acid (M) and its C-5 epimer, -L-

guluronic acid (G) residues that are covalently linked together in different sequences

(Fig.1.4). The proportions of each type of block vary depending on the source of the

alginate (Smidsrød & Skjåk-Braek 1990). Because of the diaxial links, G blocks are


18

stiffer than M blocks or alternating sequences. On the other hand, M residues are known

to provide elasticity to the gel (Augst et al. 2006a).

Figure 1.4 Alginate polysaccharide that consists of two guluronic acid and two mannuronic acid residues

with (1,4)-linkages (Augst et al. 2006a).

Alginate can be prepared with a variety of molecular weights (50-100 000KDa)

(Augst et al. 2006b) and has non-Newtonian characteristics, viscosity decreases with

increasing shear rate (Becker & Kipke 2002). The stiffness of gel network depends on the

molecular weight distribution of the polymer, the M/G ratio, and the stoichiometry of the

alginate with the multivalent cation (Augst et al. 2006b). Two G blocks of polymer chains

can be cross-linked with multivalent cations such as Ca2+

or Ba2+

by interacting with the

carboxylic groups in the polysaccharides and lead to the formation of a gel network

(Augst et al. 2006b). Calcium has been usually used as the cross-linking ion (Lim & Sun

1980). This is mainly because of its physiological and biocompatible properties that

provide a suitable condition for the cells. However, when implanted, beads made of Ca-

alginate suffer from osmotic swelling and increase permeability, destabilization, and may

result in the breakage of the beads. This is due to the fact that Ca+2

ions have a high

affinity for chelating agents such as phosphate and citrate. As a solution, beads are


19

usually coated with an additional polycation layer that strengthens the microcapsules and

adjust their permeability (Santos et al. 2009). Due to the toxicity of the polycation layer, a

second coating layer is necessary.

The most frequently applied and the most study polycation for cell encapsulation

is poly-L-lysine (PLL), which was used to construct the classical alginate-poly-L-lysine-

alginate microcapsules (APA). It has been shown that the outer layer of alginate does not

effectively hinder the immunogenic polycation layer and therefore PLL may be in contact

with the hosts’ cells (Santos et al. 2009; Clayton et al. 1991). A variety of polycations

such as poly-L-ornithine (PLO) (S K Tam et al. 2011), chitosan (Baruch & Machluf

2006), oligochitosan (T. Wang & N. He 2010), and photopolymerized biomaterials (F.

Shen et al. 2005) have been proposed for capsule construction. A study by Ponce at al.

concluded that PLL coating resulted in more stable and less immunogenic microcapsules

compared with PLO and poly-D-lysine (PDL) (Ponce et al. 2006). There are several

proposed polycations other than the ones mentioned here for construction of

microcapsules. Nevertheless, comparative in vivo studies are required to study their effect

on cell viability and functionality and compare them with the commonly used

polycations.

1.2.2.3.2 Other Polymers and Biomaterials

Hydrogels made of other biomaterials have also been investigated in cell

microencapsulation. Although none of them is as much characterized as alginate, one can

benefit from the advantages of the other biomaterials for specific applications. For


20

instance, poly(ethylene glycol) (PEG) has been used to encapsulate islet cells for diabetes

(Weber et al. 2006; Weber et al. 2007) or bone morphogenic protein (BMP) releasing

engineered fibroblasts for bone defects (Olabisi et al. 2010); hyaluronic acid (HA) has

been used to encapsulate embryonic stem cells (ESCs) or fibroblasts for tissue

engineering applications (Khademhosseini et al. 2006); HEMA copolymers have been

designed for cell encapsulation for treatment of central nervous system (CNS) diseases

(Mokrý et al. 2000).

In addition to hydrogels cross-linked by ionic interactions (such as calcium cross-

linked alginate), biomaterials made of cross-linked network of two or more polymerizable

polymers have also been studied for cell encapsulation (Santos et al. 2009). PEG and HA

functionalized with reactive groups such as methacrylates and acrylates, are one of the

mostly used for this polymerizatiom method (Khetan & J. Burdick 2009). HA is a

component of ECM and plays role in critical biological processes during wound repair.

These properties make HA an excellent candidate for cell encapsulation. It had been

demonstrated that MSCs encapsulated in HA-based hydrogels proliferate and behave

correctly (Nicodemus & Bryant 2008; Chung & J. A. Burdick 2009; Lei et al. 2011).

Cross-linked PEG hydrogel microcapsules have also been used in cell-transplantation

applications to decrease immunogenicity and enhance cell viability (King et al. 2010;

Chung & J. A. Burdick 2009). PEG-based microcapsules inhibit protein and cellular

adhesion and therefore provide lower immune response than unpurified alginate

hydrogels (Schmidt et al. 2008). However, in general PEG hydrogels present lower cell


21

viability and mechanical properties compared with alginate microcapsules (Santos et al.

2009).

Photopolymerization have become very popular in cell encapsulation strategies

considering its ability to form gels under mild physiological environment. However, HA

and PEG-based microcapsules cross-linked by photopolymerization have several

disadvantages compared with alginate-PLL-alginate microcapsules (APA) (Santos et al.

2009). First, highly reactive photoinitiator agents such as 4-

Benzoylbenzyltrimethylammonium are needed for these processes, which potentially

causes chain transfer to the molecules and proteins on cell membrane. Second, it is

necessary to fabricate temporal spherical mold as calcium alginate beads in the

fabrication process (K. H. Bae et al. 2006). Therefore, fabrication of photopolymerized

hydrogel beads is more complicated than that of APA microcapsules. Third, high

molecular weight degradation products decrease the biocompatibility of the scaffolds.

1.2.2.4 Biomaterials Modification for Bioactivity

In cell therapy strategies, the first stress the cells encounter is the lack of matrix

support. This can start even before the transplantation of cells. When cells that normally

grow in adherent cultures are forced in suspension environment, a pathway of cell death

is initiated named anoikis (Zvibel et al. 2002). Anoikis is a phenomenon occurring due to

cell detachment from the extracellular matrix (ECM) (Frisch & Ruoslahti 1997; Frisch &

Screaton 2001; Grossmann 2002; Dwayne G Stupack & David A Cheresh 2002; D G

Stupack et al. 2001; Howe et al. 2002). These days a large number of synthetic or natural


22

polymers with a variety of properties are available for cell therapy applications. One

problem is that until recently, biomaterials have been considered as an inert scaffold in

which the cells were encapsulated. Recent studies evaluate the design of novel

biomaterials to provide the cells with both a physical protective barrier and a

physiological extracellular matrix to improve the viability and functionality of the enclose

cells and to further enhance the secretion of therapeutic proteins from cells. In other

words, serving as the physiological extracellular environment (ECM) for the cells. The

search for an ideal ECM-like environment has led to design and construction of hydrogels

with incorporated cell adhesion molecules to create biomimetic biomaterials.

1.2.2.4.1 Cell-Matrix Interactions

The decision to die or live, at the cellular level, depends on the interaction of cells

with the extracellular matrix cues that trigger cell signaling pathways and promote the

cell dead or survival (Dwayne G Stupack & David A Cheresh 2002). ECM plays a critical

role in this decision-making through the action of adhesion receptors on cell surface.

Among these receptors, integrin family comprises the most numerous group and initiates

a variety of signaling pathways. Not only they play a critical role as cell adhesion

molecules, but also they are important in processes such as cell differentiation, immune

response, wound healing and hemostasis (Albelda & Buck 1990; Barczyk et al. 2010;

Dwayne G Stupack & David A Cheresh 2002; D G Stupack et al. 2001; Grossmann

2002). Integrins are heterodimeric cell adhesion molecules that consist of two non-

covalently linked subunits termed and . To date 18 and 8 subunits are known that


23

form 24 different integrins (Dwayne G Stupack & David A Cheresh 2002; Rupp & Little

2001). The combination of particular subunits determines the ligand specificity

of the integrins. Therefore, the integrins that a specific cell line express control the

repertoire of ECM components with which the cell can interact. Integrins bind to ligands

in a manner that depends on both their affinity and avidity. Different ligands or different

forms of a particular ligand can transmit different signals through a specific integrins

(Dwayne G Stupack & David A Cheresh 2002; Geiger et al. 2001). Also, a specific ligand

can be recognized by more than one integrin on the cell surface. As an instance, v

integrin binds to fibronectin, vitronectin, osteopontin, tenascin, von Willebrand factor,

thrombospondin and bone sialoprotein (Hersel et al. 2003). Also, ECM molecules such as

fibronectin interact with several integrins (Plow et al. 2000). This will increase the level

of complexity to cellular response to the ECM.

Integrins directly activate survival pathways via the PI-3 kinase and MAPK

pathways. On the other hand, elevated expression of integrins in the absence of

extracellular cues results in apoptotic cell death under otherwise permissive growth

conditions. Thus, integrins play a crucial dual role coordinating survival or death

responses as a function of ECM composition.

Figure 1.5 Immobilized ligands lead to cell adhesion and survival (Hersel et al. 2003).


24

Integrins govern cellular adhesion and shape that play critical roles in the response

of cells to the survival factors (Ingber 1992; Meredith et al. 1993). Physical association of

several growth factors receptors with integrins explain the integrin requirement for

growth factor signaling (Dwayne G Stupack & David A Cheresh 2002; Borges et al.

2000; Eliceiri 2001). Also, integrins directly induce cell-signaling pathways by ligation-

dependent recruitment of non-receptor tyrosine kinases from focal adhesion kinase (FAK)

and Src families, which result in the activation of several signaling pathways. The

consequent signals, especially the ones through the mitogen activated protein kinase

(MAPK) and phosphoinositide 3-kinase (PI3K) are crucial for the cyclin-dependent

kinases (CDK) and cell cycle progression (Schwartz & Assoian 2001).

Integrins prevent apoptosis by maintaining cell’s normal function through intrinsic

death pathways (stress pathway or mitochondrial pathway) or extrinsic pathway (Death

receptors). Ligation of integrins 5 v vleads to increased

expression of Bcl-2, a member of apoptosis regulator proteins, and increased resistance to

serum withdrawal (Matter & Ruoslahti 2001).

Strong integrin interactions with the ECM ligand may convince the cell that it is

well suited to its environment regardless of the existence of proapoptotic signals. So if the

proapoptotic insults should be overcome, the cells can be rescued rather than lost or

replaced. Conversely, the cells that lack the expression of integrins that interact with the

ECM molecules, are more susceptible to stress and lose viability more rapidly in

proapoptotic conditions (Dwayne G Stupack & David A Cheresh 2002).


25

1.2.2.4.2 Modification with Cell-Adhesive Molecules

Several studies have addressed the significance of selected ECM proteins such as

fibrinogen or fibronectin in enhancing survival signals demonstrating different results

based on the cell type in question (Grossmann 2002; Fukai et al. 1998; Dwayne G

Stupack & David A Cheresh 2002; D G Stupack et al. 2001; Karoubi et al. 2009). In a

study by Karoubi et al., human marrow stromal cells were singularly encapsulated in

agarose microcapsules incorporated with fibrinogen and fibronectin molecules with the

aim of enhancing cell-matrix interactions. Cells in modified microcapsules demonstrated

enhanced viability and greater metabolic activity due to re-introduction of cell-matrix

interactions. It was concluded that the attenuation of anoikis in this cell-matrix system is

by cell integrin-matrix interaction and activation of MAPK/ERK signaling pathway. In

vivo studies reveal that the single-cell engineered microcapsules enhanced the retention

of marrow stromal cells in the rat hindlimb (Karoubi et al. 2009).

Using cell recognition motifs as small as immobilized peptides rather than full-

length proteins to design biomimetic biomaterials bear several advantages in medical

applications. Proteins need to be isolated from other organisms and purified. Therefore,

they may increase the chance of infection and undesired immune response. Additionally,

only a part of the proteins have a proper orientation for cell adhesion (Elbert & Hubbell

2001; Hersel et al. 2003). On the other hand, peptides demonstrate higher stability during

sterilization processes, pH variation, heat treatment, storage, as well as cost effectiveness.

Also, because of their smaller size, they can be packed with higher density on

biomaterials (Hersel et al. 2003; Ruoslahti 1996; Neff et al. 1998; Ito et al. 1991; Boxus


26

et al. 1998). Additionally, while full-length ECM proteins contain many different cell

recognition motifs, peptides represent just one motif. Therefore, peptides address one

particular cell adhesion receptors (Hersel et al. 2003).

The search for an ideal ECM-like microenvironment has led to design and

construction of hydrogels that incorporate different integrin-mediated cell adhesion

sequences including RGD, IKVAV, LRE, YIGSR, PDSGR, IKLLI (Santos et al. 2009;

Weber et al. 2007; Zustiak et al. 2010; Horák et al. 2011). The most widely employed

peptide sequence is arginine-glycine-aspartic acid tri-peptide (RGD) first identified as a

minimal essential cell adhesion peptide sequence in fibronectin (Pierschbacher &

Ruoslahti 1984). Since then, the cell adhesive RGD sequence was found in many other

ECM proteins including, fibrinogen, vitronectin, collagen, von Willebrand factor,

osteopontin, bone sialoprotein, and laminin (Pfaff 1997). RGD is the most widely studied

and employed peptide for cell adhesion. This is due to its widespread distribution

throughout the organism, its ability to address several cell adhesion receptors, and its

impact on cell attachment and survival (Hersel et al. 2003). About half of the cell surface

integrins were shown to bind to ECM molecules in a RGD dependent manner:

vv3vvv (Pfaff 1997).

Although there are other important cell adhesion motifs and RGD is not the

universal cell recognition motif, it is nevertheless unique because of its broad distribution

and usage (Hersel et al. 2003).


27

Figure 1.6 The RGD sequence and its molecular formula (Hersel et al. 2003).

Coupling of RGD peptide to alginate hydrogel has been extensively studies by

Mooney et al. (Jon A Rowley & David J Mooney 2002; Comisar et al. 2007; Comisar et

al. 2006; M.-S. Bae et al. 2007; J A Rowley et al. 1999). RGD was chemically cross-

linked to alginate utilizing aqueous carbodiimide chemistry. A water soluble

carbodiimide, 1-ethyl-(dimethylaminopropyl) carbodiimide (EDC) (Hermanson 1996),

was used to form amide bond between amine containing molecules and carboxylic

functional groups on alginate backbone. The reactive EDC-intermediate was stabilized

against a competing hydrolysis reaction using the co-reactant N-hydroxysulfosuccinimide

(sulfo-NHS) (Hermanson 1996). This raised the efficiency of amide bond formation.

RGD was then conjugated to the alginate backbone using the terminal amine of the

peptide (Figure 1.7) (J A Rowley et al. 1999).


28

Figure 1.7 Peptide coupling to alginate backbone (J A Rowley et al. 1999). Amide bond is formed by the

cabodiimide through the carboxyl functional group of alginate and N-terminal amide of the RGD peptide.

Functionalization of 3D alginate microcapsules with RGD has been recently

transferred to the field of cell microencapsulation, significantly improving the features of

this technology. Orive et al. have shown that integrin binding to RGD cross-linked to

alginate act as additional cross-linkage within the alginate matrix enhancing the

mechanical stability of the microcapsule system (Figure 1.8). Additionally, the RGD-

alginate microcapsules provide cell adhesion for the enclosed MSCs, which result in a

more physiological environment for the enclosed cells that improves their viability and

cell behavior. In vivo studies confirmed the long-term functionality and drug release,

resulting in sustained erythropoietin delivery during 300 days without

immunosuppressive drugs (Orive et al. 2009).


29

Figure 1.8 RGD modification provides attachment site for enclosed cells (Orive et al. 2009).

A study by Markusen et al. also demonstrates that RGD-alginate microcapsules

substantially retain the viability of the enclosed cells. GRGDY-alginate microcapsules

where shown to encourage the attachment and elongation of the enclosed MSCs and

result in a dense network of cells that is crucial for tissue substitution applications

(Markusen et al. 2006). It is well known that cells in monolayer cultures differ from their

counterparts in 3D suspension culture in terms of cell morphology, proliferation and gene

expression. Little is known about the phenotype and gene expression of MSCs enclosed

in alginate-based microcapsules. A recent study by Duggal et al. compares the MSC

morphology and gene expression when cultures in monolayer versus RGD-alginate 3D

microcapsules (Duggal et al. 2009). MSCs changed from the fibroblastic morphology in

2D to small compact shape when embedded in RGD-alginate microcapsules. The viability

was maintained at a high value during the in vitro experiments. However, MSCs ceased to

proliferate when grown in 3D microcapsules. It was also demonstrated that MSCs

retained expression of integrins that are known to bind the RGD peptide sequence. MSCs

cultured in the monolayer culture showed high level expression of v and


30

moderate expression of . After 7 days of culture in RGD-alginate microcapsules, some

if these integrins were slightly down regulated, while the expression of the others was

retained. It was demonstrated in this study that the gene expression in cells in RGD-

alginate was different both from the cells grown in monolayer culture and from isolated

uncultured MSCs, but more similar to monolayer cultures (Duggal et al. 2009).

The literature reviewed in this section shows that recombinant cell encapsulation

can be applied as a cell therapy strategy for hemophilia treatment. Previous studies also

demonstrate that hydrogel microcapsules modified with bioactive molecules, either

proteins or peptides, can enhance the viability and functionality of the enclosed cells.

More research in this area is crucial to design and construct biocompatible and bioactive

microcapsules to enhance the cell viability and FIX secretion from the enclosed cells as a

potential treatment for hemophilia B.


31

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41

CHAPTER 2: OBJECTIVES AND CONTRIBUTIONS TO ARTICLES

2.1 OBJECTIVES

From the discussion in Chapter 1, it is clear that despite a variety of research studies

on gene therapy for hemophilia treatment, development of a safe and cost-effective

treatment for hemophilia is desired. Considering the current advances in both biomaterial

science and stem cell technologies, design and construction of biocompatible cell-loaded

microcapsules for sustained and therapeutic in vivo release of FIX can potentially serve as

an effective treatment for this disease. Also, it is evident from the literature review in

Chapter 1 that in spite of some promising results in animal studies, improvement of

viability and functionality of the enclosed cells in encapsulation devices are still major

challenges in application of cell encapsulation for hemophilia. In this regard, bioactive

microcapsules with advantages over bioinert microcapsules may have superior effect on

viability and functionality of the enclosed cells.

The overall objective of this work is design and construction of biomimetic

alginate-based microcapsules for enhanced cell viability and FIX secretion from the

enclosed FIX-engineered mesenchymal stem cells. In this regard, fibrinogen and

fibronectin as two well-known cell-adhesive proteins and RGD as a widely studied cell-

adhesive peptide are used to modify the alginate matrix. There are three main parts to this

research, design and construction of (1) fibrinogen-alginate, (2) RGD-alginate, and (3)

fibronectin-alginate microcapsules.


42

In the first part, the objectives are: to assess the viability and FIX secretion level of

umbilical cord blood-derived (CB) MSCs in three-dimensional alginate microcapsules; to

investigate whether alginate microcapsules can be manipulated to increase viability,

proliferation and FIX secretion by incorporating fibrinogen as a cell-adhesion protein; and

to induce differentiation of CB MSCs in fibrinogen-alginate microcapsules into osteogenic,

chondrogenic and adipogenic lineages in order to examine the effect of differentiation on

cell viability and FIX secretion.

In the second part, the objectives are: to investigate whether we can manipulate the

alginate microcapsules by incorporating RGD peptide to provide the required cell matrix

signaling to increase cell viability and FIX secretion from the encapsulated cells; to

compare effect of RGD-coupled alginate with the effect of the previously designed

fibrinogen-alginate microcapsules on viability, proliferation and FIX secretion of the

encapsulated MSC.

In the third part, the objectives are: to assess whether the incorporation of

fibrinogen within alginate matrix can manipulate the hydrogel microcapsules for enhanced

viability and FIX secretion level of umbilical cord blood-derived (CB) MSC; to investigate

how the different concentrations of fibronectin can affect cell viability, proliferation and

FIX secretion; to induce differentiation of the encapsulated CB MSC in fibronectin-

alginate microcapsules into osteogenic, chondrogenic and adipogenic lineages in order to

examine the effect of differentiation status of the encapsulated cells on cell viability and

FIX secretion.


43

2.2 CONTRIBUTIONS TO ARTICLES

This thesis contains three articles (Chapters 3-5). My contributions involve the design

and performance of the three projects, including literature searches, experiments and data

analysis. I wrote the first draft of all three papers and the initial responses to the questions

and comments of the reviewers. Then, I worked with my supervisor on the later drafts of

the papers. For all the papers, Megan Dodd transduced the human cord blood-derived

mesenchymal stem cells for FIX secretion.

The first paper (chapter 2) is accepted at the Journal of Tissue Engineering. The

second paper (chapter 3) and third paper (chapter 4) are at the final steps of revision and

they are to be submitted to the Journal of Artificial cell, Nanomedicine and Biotechnology

and Journal of Tissue Engineering, Part A. I have orally presented the first paper and

second paper at the Canadian Biomaterial Society Conference (Vancouver 2011), and at

the World Biomaterial Congress, (Chengdu 2012), respectively. I will orally present the

third paper at the XX International Conference on Bioencapsulation (Orillia 2012).


44


MESENCHYMAL STEM CELLS IN FIBRINOGEN-ALGINATE

MICROCAPSULES ENHANCES CELL VIABILITY AND FIX SECRETION

Authors: Bahareh Sayyar, Megan Dodd, Jianping Wen, Shirley Ma, Leah Marquez-

Curtis, Anna Janowska-Wieczorek, Gonzalo Hortelano

Publication Information: Journal of Tissue Engineering

Acceptance Date: August 17, 2012

Working Hypothesis:

Improved cell viability and FIX secretion from the encapsulated MSCs may be obtained

by decorating the core of alginate microcapsules with fibrinogen molecules.

Differentiation induction of the encapsulated cells through osteogenic, chondrogenic, or

adipogenic media may affect the viability and functionality of the encapsulated cells.


45

Encapsulation of FIX-Engineered Human Mesenchymal Stem Cells in Fibrinogen-

Alginate Microcapsules Enhances Cell Viability and FIX secretion

Bahareh Sayyar1, Megan Dodd

1, Jianping Wen

2, Shirley Ma

2, Leah Marquez-Curtis

3,

Anna Janowska-Wieczorek3,4

, Gonzalo Hortelano1,2*

1 School of Biomedical Engineering, McMaster University, Canada

2 Department of Pathology & Molecular Medicine, McMaster University, Canada

3Research & Development, Canadian Blood Services, Canada

4Department of Medicine, University of Alberta, Canada

Corresponding author: Gonzalo Hortelano

School of Biomedical Engineering

McMaster University

Hamilton, ON Canada L8S 4K1

Ph: +1 (905) 525-9140 ext. 22739

Fax: +1 (905) 521-2613 Email: [email protected]

mailto:[email protected]


46

Abstract

Cell microencapsulation holds a significant promise as a strategy for cellular

therapy; however, inadequate survival and functionality of the enclosed cells are among

the most critical factors limiting its application in hemophilia treatment. Here we describe

the use of alginate-based microcapsules to enhance the viability and transgene secretion

of human cord blood-derived (CB) mesenchymal stem cells (MSCs) in three-dimensional

cultures. Given the positive effects of extracellular matrix molecules on MSC growth, we

tested whether fibrinogen-supplemented alginate microcapsules can improve the

efficiency of encapsulated FIX-engineered MSCs as a treatment of hemophilia B. We

found that fibrinogen-supplemented alginate microcapsules significantly enhanced the

viability and proliferation of FIX-engineered MSCs, which resulted in improved FIX

secretion in comparison to MSCs in non-supplemented microcapsules. Differentiation

studies showed the osteogenic (but not chondrogenic or adipogenic) differentiation

capability of FIX-engineered CB MSCs and their efficient FIX secretion while

encapsulated in fibrinogen-supplemented alginate microcapsules. Thus, the use of

recombinant MSCs encapsulated in fibrinogen-modified microcapsules may be of

practical use in the treatment of hemophilia or other protein deficiency diseases.

KEYWORDS

Mesenchymal stem cells, hemophilia, alginate, fibrinogen, cell encapsulation


47

1. Introduction

Hemophilia B is an X-linked bleeding disorder caused by human factor IX (FIX)

deficiency that occurs in 1 in 30,000 males (Sadler & Davie 1987a),(Bolton-Maggs & K.

J. Pasi 2003a). In the case of severe hemophilia, current treatment involves the life-long

and costly infusion of recombinant or plasma-derived FIX protein (A Gater et al. 2011).

Gene therapy offers an attractive alternative to the current treatment because FIX

expression is loosely regulated and tissue-specific expression of a transgene is not

required. The supply of as little as 1% of the physiological concentration of FIX would

have clinical benefits (Manno et al. 2003). Currently, successful gene therapy methods

involve direct injection of viral vectors for FIX delivery but there are still some associated

safety concerns and development of an alternative method of FIX delivery is desirable

(Nathwani et al. 2011c; Marshall 2001a; Check 2003a). Here we evaluate genetically

engineered allogeneic cells for their potential use in therapies for hemophilia B.

Implantation of cells enclosed in biocompatible, semi-permeable microcapsules

leads to continuous delivery of therapeutic proteins and protects cells from the host

immune response (Orive et al. 2003b; Orive et al. 2006a; Thakur et al. 2010a; G

Hortelano et al. 1996b; Wen et al. 2007a; Wen et al. 2006b; G Hortelano et al. 2001b).

The host genome is not modified and most of the microcapsules can be removed should it

become necessary to reverse the treatment. As an additional safety measure, recombinant

cells may be thoroughly characterized for unwanted genetic rearrangements before

encapsulation. To overcome the challenge of engineering cells from each individual, a


48

universal cell line can be engineered and encapsulated in biocompatible microcapsules to

be implanted in different patients.

We previously described the use of alginate microcapsules enclosing recombinant

fibroblasts, C2C12, or G8 myoblasts to deliver hFIX in mice (G Hortelano et al. 1996b;

Wen et al. 2007a; Wen et al. 2006b; G Hortelano et al. 2001b; G Hortelano et al. 1999; G

Hortelano & P. L. Chang 2000). We also demonstrated that transplanted encapsulated

cells play a key role in the immune response generated in the host against the

transgene.(Wen et al. 2006b) It has been indicated that fetal myoblasts do not elicit the

strong immune response to FIX seen in transformed myoblasts and therefore are more

suitable for cell therapy strategies (Wen et al. 2006b). Transplantation of mesenchymal

stem cells (MSCs) may also be a feasible strategy for the treatment of hemophilia B.

MSCs are easy to culture, can be genetically manipulated to secrete bioactive molecules,

are susceptible to molecules that modify their natural behavior, and have an

immunosuppressive effect on immune cells involved in alloantigen recognition and

elimination (M. Liu & Han 2008b),(Frisch & Ruoslahti 1997).

Biomaterial surfaces mimic the local microenvironment of cells by regulating cell

attachment, viability, proliferation, migration, differentiation, and secretion of proteins.

Alginate, the most commonly used biomaterial for cell encapsulation, does not provide

the sufficient cues for cell-matrix interactions (Zvibel et al. 2002; Dwayne G Stupack &

David A Cheresh 2002; Valentijn et al. 2004; Grossmann 2002). During cell

encapsulation, anchorage-dependent MSCs encounter a lack of attachment and support.

Current research in biomaterials and cell-biomaterial interactions focuses on


49

compensating for this deficiency by providing the encapsulated cells with a

microenvironment that more closely mimics their natural environment. One way to

improve performance of the existing biomaterials is surface modification using bioactive

molecules, such as native full-length extracellular matrix (ECM) proteins or the short-

sequence peptides derived from them (Shin et al. 2003a). Optimally modified

biomaterials would mimic the natural physiological environment of the encapsulated cells

(“biomimetic”) and be readily available off the shelf. Of the myriad of potential

biomaterials available, several studies have addressed the significance of fibrinogen as a

cell-adhesion protein in mediating cell survival, and different results have been reported,

depending on the cell type in question (Dwayne G Stupack & David A Cheresh 2002;

Karoubi et al. 2009b; D G Stupack et al. 2001; Whitlock et al. 2000a). The focus of this

work is to design and construct novel biomimetic microcapsules and to monitor viability,

FIX secretion and differentiation of the enclosed CB-MSCs in vitro.

The objectives of the present study are three-fold: i) to assess the viability and FIX

secretion level of umbilical cord blood-derived (CB) MSCs in three-dimensional alginate

microcapsules; ii) to investigate whether alginate microcapsules can be manipulated to

increase viability, proliferation and FIX secretion by incorporating fibrinogen as a cell-

adhesion protein; and iii) to induce differentiation of the encapsulated CB MSCs in

fibrinogen-supplemented alginate microcapsules into osteogenic, chondrogenic and

adipogenic lineages in order to examine its effect on cell viability and FIX secretion.


50

2. Materials and methods

2.1 Cell culture

Umbilical cord blood was obtained after delivery with the mothers’ informed

consent in accordance with the guidelines of the University of Alberta Health Research

Ethics Board. Light density mononuclear cells (MNC) were separated by Percoll density

gradient centrifugation (Amersham Biosciences, Uppsala, Sweden) and cultured in

Iscove’s Modified Dulbecco’s Media (IMDM; Invitrogen, Burlington, ON, Canada)

supplemented with 10% fetal bovine serum and streptomycin (100 µg/ml) at 37°C in 5%

CO2. After 24h, non-adherent cells were removed and complete medium was replaced, as

described previously (Son et al. 2006). MSCs were used in experiments before reaching

passage 6.

2.2 Engineering of MSCs

The PLVX-Puro vector DNA (Clontech, Mountain View, CA, USA) was

engineered with traditional restriction enzyme techniques to generate a FIX-expressing

lentiviral DNA construct with CMV promoter. Viral particles were generated with the

Lenti-X Expression System (Clontech) according to the manufacturer’s protocol. Briefly,

LentiX 293T cells were transfected with the 4th generation VSV-G packaging DNA and

PLVX-FIXI expression plasmid using Xfect transfection reagent to generate viable virus

particles. Two rounds of freshly produced viral supernatant were used to transduce CB

MSCs (passage 4) for 24 h at a MOI of approximately 20. Transduced cells were selected

with puromycin (3 μg/ml) (Mountain View, CA, USA).


51

2.3 Cell encapsulation

MVG ultrapure alginate was purchased from FMC, BioPolymer (Philadelphia,

PA, USA). FIX-engineered CB MSCs were suspended in a fibrinogen-supplemented and

non-supplemented (control) alginate solution (1.56% alginate) at a concentration of 3x106

cells/ml. Microencapsulation was performed with an electrostatic encapsulator (Nisco

Engineering Inc., Zurich, Switzerland) as previously described(P. L. Chang et al. 1994a).

Briefly, cell mixture was pumped through the electrostatic encapsulator (voltage: 7kV) at

the flow rate of 0.9 ml/min into a vial containing 1.1% CaCl2. Cell-loaded beads were

then washed with the saline solution and cross-linked with poly-L-lysine and with an

outer layer of non-supplemented alginate. Before the encapsulation, human plasma

fibrinogen (Sigma Aldrich, Oakville, ON, Canada) was added to the alginate core as

necessary at a concentration of 750 µg/ml of alginate solution.

For the monolayer studies, non-supplemented (1.56% alginate) or fibrinogen-

supplemented (750 mg fibrinogen/ml of 1.56% alginate) alginate mixture was placed over

the sterilized cover slips. CaCl2 was dropped on the cover slips in order to gel the

alginate. Coated cover slips were kept under the fume hood over night and were then

seeded with CB-MSC and incubated overnight under normal tissue culture conditions.

2.4 Differentiation of human MSCs

For induction of osteogenic, chondrogenic, or adipogenic differentiation,

encapsulated MSCs were cultured in StemPro Osteogenic, StemPro Chondrogenic, or


52

StemPro Adipogenic differentiation medium (GIBCO Invitrogen, Burlington, ON,

Canada), respectively and with appropriate supplements.

Non-encapsulated cells were grown in differentiation media in monolayer cultures

and stained for differentiation markers. At week 3-post osteogenic induction and at week

2-post chondrogenic and adipogenic induction, cells were washed with phosphate

buffered saline (PBS), fixed in 4% paraformaldehyde for 10 min and washed. Cells were

stained with Alizarin Red, Alcian Blue, and Oil Red O dyes (Sigma Aldrich) for detection

of calcium deposits, proteoglycans, and fat vacuoles as an indication of osteogenic,

chondrogenic, and adipogenic differentiation, respectively. Cells were visualized under an

inverted light microscope (Leica DM IL, Leica Microsystems, Richmond Hill, ON,

Canada).

2.5 Assessment of viability and proliferation of encapsulated cells

Viability of encapsulated MSCs was assessed using the trypan blue exclusion

assay(Son et al. 2006). Proliferation of encapsulated MSCs was assessed by MTT assay

(Sigma-Aldrich, Oakville, ON, Canada) according to the manufacturer’s instruction

adapted to encapsulated cells. Briefly, 100 μl of microcapsules were incubated with 10 μl

MTT reagent in 96-well plate for 3 hours at 37o C. After 3 hours, when the intracellular

punctate (purple precipitate) was clearly visible under the microscope, the resultant

formazan was dissolved in 100 μl dimethyl sulphoxide (DMSO, BDH Inc. Toronto, ON,

Canada). The plate was incubated in the dark for 3 hours at room temperature and


53

absorbance measured at 562 nm (EL 808, ultra microplate reader, BIO-TEK Instruments

Inc., Winooski, VT, USA).

2.6 F-actin cytoskeleton staining of encapsulated cells

Encapsulated cells (100 μl of capsules) were fixed in 4% paraformaldehyde. As

indicated in manufacturer’s protocol, cells were then washed in pre-warmed PBS and

permeabilized by incubation in 0.1% Triton X-100 in PBS for 10 minutes, followed by

two washes in PBS. Cells were stained with Alexa Fluor 633 phalloidin (15 μl methanolic

stock solution in 200 μl PBS, for 30 min in the dark) (Molecular Probes Invitrogen,

Burlington, ON, Canada) containing 1% bovine serum albumin to reduce nonspecific

background. A sample was analyzed by inverted confocal microscopy (Zeiss 510, Carl

Zeiss Inc., Toronto, Ontario, Canada).

2.7 Transmission electron microscopy and scanning electron microscopy

All transmission electron microscopy (TEM) and scanning electron microscopy

(SEM) studies were done at the electron microscopy facility, McMaster Children’s

Hospital (Hamilton, ON, Canada). Encapsulated cells were fixed with 2% glutaraldehyde

(2% v/v) in 0.1M sodium cacodylate buffer (pH 7.4). The samples were rinsed 2X in

buffer solution, then post-fixed in 1% osmium tetroxide in 0.1M sodium cacodylate

buffer for 1 hour and dehydrated through a graded ethanol (EtOH) series (50% to 100%).

For TEM analysis, the final dehydration of the TEM samples was done in 100%

propylene oxide (PO). Infiltration with Spurr's resin was done through a graded series of


54

PO:Spurr’s 2:1, 1:1, 1:2, 0:1 with rotation of the samples in between solution changes.

The samples were transferred to embedding moulds which were then filled with fresh

100% Spurr's resin and polymerized overnight in a 60°C oven. Sections were cut on a

Leica UCT ultramicrotome (Leica Microsystems, Richmond Hill, ON, Canada) and

picked up onto copper grids. Sections were post-stained with uranyl acetate and lead

citrate and viewed in a JEOL JEM 1200 EX TEMSCAN transmission electron

microscope (JEOL, Peabody, MA, USA) at an accelerating voltage of 80kV. For SEM

analysis, after dehydration in 100% EtOH some capsules were frozen, fractured, and

placed back into 100% EtOH to thaw. Samples were then critical-point dried, mounted

onto SEM stubs, sputter-coated with gold, and viewed in a Tescan Vega II LSU scanning

electron microscope (Tescan USA, Cranberry Twp., PA) at 20kV.

2.8 FIX ELISA assay

Human FIX antigen in culture media was quantified by an ELISA assay (Affinity

Biologicals Inc., Ancaster, ON, Canada), as previously described(Wen et al. 2006b).

2.9 Animal studies

All animal procedures were conducted in accordance with the Animal Ethics

Guidelines of McMaster University. C57BL/6 mice were purchased from Charles River

(Montreal, QC, Canada) and maintained at the Central Animal Facility, McMaster

University (Hamilton, ON, Canada). Mice were anaesthetized with isofluorane (Bimeda-

MTC, Animal Health Inc., Richmond Hill, ON, Canada) in a small animal anaesthetic


55

machine (Med-Vet Anaesthetic System Inc., Toronto, ON, Canada). Each mouse received

2 ml of microcapsules intraperitoneally using a G18 catheter.

2.10 Statistical data analysis

Analysis of variance (ANOVA) was carried out to determine whether significant

differences existed in the groups of data. Student’s t-test or Tukey HSD were conducted

as post hoc tests to compare the pairs of data. Differences were considered significant

when p<0.05. Data are expressed as means ± SD.

3. Results

3.1 Determining cell-matrix interactions in monolayer

In order to assess the effect of fibrinogen on the morphology and proliferation of

MSCs, 3x106 cells were cultured in monolayer overnight on cover slips coated with non-

supplemented alginate (control) or fibrinogen-supplemented alginate. The presence of

fibrinogen resulted in dramatic enhanced proliferation of MSCs (Figure 1).


56

Figure 1. Effect of fibrinogen-supplemented alginate on proliferation of FIX-engineered CB MSCs. Light

microscopy images (x10) of MSC grown in monolayer at 24 h post culture on: (a) non-supplemented

alginate-coated cover slip, (b) Fibrinogen-supplemented alginate-coated cover slip.

3.2 Assessment of MSC viability and proliferation in microcapsules

To determine the proliferation and FIX secretion of MSCs in fibrinogen-

supplemented and non-supplemented (control) alginate microcapsules, encapsulated cells

were cultured in vitro for 28 days. MTT assay confirmed that fibrinogen significantly

increases the viability of encapsulated cells over control capsules (Figure 2(a)). Trypan

blue assay showed no significant difference on day 1 (82% in the control group and 79%

in the fibrinogen-supplemented group). Viability dropped to 55% by day 28 in control

microcapsules but remained high (72%) in fibrinogen microcapsules. Additionally, an

MTT assay confirmed that cell proliferation is significantly higher in fibrinogen-alginate

microcapsules than in alginate microcapsules throughout the experiment (Figure 2(b)).

MSC proliferation was significantly higher in the presence of fibrinogen throughout the

experiment and was 35% higher on day 28 (Figure 2(b)).


57

Figure 2. Viability and proliferation of CB MSCs. (a) The effect of fibrinogen-supplemented microcapsules

on viability of encapsulated cells. The ratio of viable cells in fibrinogen-supplemented microcapsules to the

viable cells in non-supplemented microcapsules was calculated using the MTT assay. (b) Comparison of

MSC proliferation in non-supplemented and fibrinogen-supplemented microcapsules. The ratio of viable

cells per viable cells at day 1 was calculated using the MTT assay. Data are means ± SD, n=3, Student’s t-

test. *(A) Significant difference from 1.000, P<0.05; *(B) Significant difference non-supplemented

microcapsules vs. fibrinogen-supplemented microcapsules on each day, P<0.05.


58

3.3 FIX secretion from encapsulated cells

Aside from stable proliferation, a sustained FIX secretion is of paramount

importance. Consistent with the significantly higher viability and proliferation of MSCs,

incorporation of fibrinogen in the alginate matrix resulted in higher FIX secretion from

the encapsulated MSCs throughout the experiment, as measured by ELISA. The average

FIX secretion from fibrinogen-supplemented device was above 4000 ng/ml (capsules/24

h) during the first two weeks of in vitro growth (Figure 3). In agreement with the trend in

cell proliferation (Figure 2), FIX secretion dropped to above 2000 ng/ml (capsules/24 h)

in cells in fibrinogen microcapsules and to ~1000 ng/ml (capsules/24 h) in control

microcapsules (Figure 3).

Figure 3. FIX secretion by CB MSCs. Comparison of FIX secretion from MSCs in non-supplemented and

fibrinogen-supplemented microcapsules. FIX secretion was measured using ELISA assay and reported as

the amount of FIX (ng) secreted from 1 ml of microcapsules in 24 h. Data are means ± SD, n=4. p<0.05,

Student’s t-test. *Significant difference, non-supplemented microcapsules vs. fibrinogen-supplemented

microcapsules on each day.


59

3.4 Cell-matrix analysis

For an in-depth, three-dimensional look at the cell-matrix interaction, we

examined encapsulated MSCs using confocal microscopy, TEM, and SEM (Figure 4). As

detected by TEM, in fibrinogen cells clearly demonstrated the presence of filopia-like

membrane extensions into the surrounding matrix (Figure 4(a) and (b)), which were

lacking in control microcapsules (Figure 4(c) and (d)). However, SEM and confocal

analysis did not reveal significant differences between both groups in terms of

morphology or F-actin staining (Figure 4(e) to (h) and Figure 4(i) to (l)). By confocal

microscopy both groups of microcapsules lacked detectable patterns or alignments of

cellular filaments (Figure 4(i) to (l)). Further, SEM images did not show any obvious

differences in cell morphology between both types of microcapsules (Figure 4(e) to (h)).


60

Figure 4. (a-d) Transmission electron microscopy images of CB MSCs encapsulated in fibrinogen-

supplemented alginate microcapsules (a, b) or non-supplemented alginate microcapsules (c, d). (e-h)

Scanning electron microscopy images of CB MSCs encapsulated in fibrinogen-supplemented alginate

microcapsules (e, f) or non-supplemented alginate microcapsules (g, h). (i-l) F-actin staining of CB MSCs

encapsulated in fibrinogen-supplemented alginate microcapsules (i, j) or non-supplemented alginate

microcapsules (k, l). Cells were fixed 7 days after encapsulation and processed for electron and confocal

microscopy. Bars indicate 2 microns.


61

3.5 Effect of differentiation induction on encapsulated cells viability, proliferation, and

FIX secretion

To investigate the effect of viral transduction on the differentiation potential of

MSCs, engineered cells secreting FIX were grown in monolayer cultures and induced to

differentiate into osteogenic, chondrogenic and adipogenic lineages. FIX-engineered cells

successfully differentiated into osteocytes within 3 weeks (Figure 5(a)) and chondrocytes

within 2 weeks (Figure 5(b)), but not adipocytes (Figure 5(c)). The lack of adipogenic

differentiation is consistent with previous reports of CB MSCs (Rebelatto et al. 2008). A

comparable differentiation potential was detected in non-transduced MSCs. Our results

show that transduction of MSCs with lentivirus did not affect its differentiation potential.


62

Figure 5. Differentiation potential of FIX-engineered CB MSCs. (a) Osteogenic differentiation, (b)

Chondrogenic differentiation, (c) Adipogenic differentiation.

Further, the effect of the differentiation induction on cell viability and

proliferation was assessed. MSCs in fibrinogen microcapsules were cultured in one of

adipogenic, chondrogenic or osteogenic differentiation medium. Some microcapsules

were cultured in control basal medium. The total number of viable cells in osteogenic,

chondrogenic and adipogenic differentiation media were normalized with respect to the

total number of viable cells in basal medium (Figure 6(a)). The viability of cells grown in

osteogenic medium was not significantly different from the viability of cells grown in

basal medium, and significantly higher than cells grown in chondrogenic and adipogenic

media. A similar trend was also observed with cellular proliferation in the different media

during 28 days in vitro (Figure 6(b)). The low viability and proliferation of encapsulated

cells grown in chondrogenic and adipogenic media suggest the inability of the proposed

fibrinogen-alginate microcapsule system to support chondrogenic and adipogenic

differentiation.


63

Figure 6. Viability and proliferation of CB MSCs. (a) MSC viability in fibrinogen-supplemented

microcapsules cultured in differentiation media normalized to the viability of cells grown in basal media.

(b) Comparison of MSC proliferation in fibrinogen-supplemented microcapsules grown in basal and

differentiation media. Data are means ± SD, n=3. The ratio of viable cells per viable cells at day 1 was

calculated using the MTT assay. *Significant difference test-basal vs. test-chondrogenic and test-

adipogenic, =0.05, Tukey HSD. **Significant difference test-osteogenic vs. test-chondrogenic and test-

adipogenic, =0.05, Tukey HSD. ***Significant difference from 1.000, p<0.05, Student’s t-test. .


64

The effect of differentiation induction on FIX secretion was also investigated.

During the first two weeks of culture, FIX secretion from encapsulated cells cultured in

basal media was significantly higher than the secretion from cells grown in osteogenic,

chondrogenic or adipogenic media (Figure 7). By the third week, FIX secretion from the

cells grown in basal and osteogenic media was significantly higher than the secretion

from cells grown in chondrogenic or adipogenic media, consistent with the differentiation

time of MSCs into osteoblast (21 days, osteogenic differentiation protocol, GIBCO

Invitrogen). The low FIX secretion from cells grown in chondrogenic and adipogenic

media was consistent with the low viability and proliferation observed for the cells grown

in these conditions.

Figure 7. FIX secretion of CB MSCs. Comparison of FIX secretion from the MSCs in fibrinogen-

supplemented microcapsules grown in differentiation media (Data are means ± SD, n=4). FIX secretion was

measured using ELISA. =0.05, Tukey HSD. *Significant difference Fibrinogen-alginate (basal) vs. all

other conditions. **Significant difference Fibrinogen -alginate (basal) vs. Fibrinogen -alginate


65

(chondrogenic) and (adipogenic). ***Significant difference Fibrinogen -alginate (osteogenic) vs.

Fibrinogen -alginate (chondrogenic) and (adipogenic).

3.6 Biocompatibility of fibrinogen-supplemented alginate microcapsules in vivo

To assess the biocompatibility of the fibrinogen-supplemented microcapsules,

fibrinogen-supplemented and non-supplemented alginate microcapsules were injected

without cells intraperitoneally in C57BL/6 mice. All mice were sacrificed on day 7 post-

treatment, and implanted capsules were retrieved and analyzed by inverted light

microscopy (Figure 8). The biocompatibility of both types of microcapsules looked

similar. There was no obvious overgrowth around the fibrinogen-supplemented

microcapsules, suggesting good biocompatibility.

Figure 8. Empty capsules retrieved from mice after 7 days in vivo. (a) non-supplemented alginate-PLL-

alginate microcapsules. (b) Fibrinogen-supplemented alginate-PLL-alginate microcapsules (no significant

overgrowth around microcapsules).


66

To monitor fibrinogen leakage, 2 ml microcapsules were suspended in 10 ml PBS

and regular supernatant samples were analyzed for evidence of fibrinogen using UV

spectroscopy at 280 nm. As observed by the UV spectroscopy, fibrinogen leakage was

undetectable throughout the experiment (with a minimum level of detection of 0.5µg/ml)

(data not shown). This is consistent with the good biocompatibility observed in mice

(Figure 8).

4. Discussion

The purpose of this study was to investigate the viability, proliferation and FIX

secretion of engineered CB MSCs encapsulated in alginate-fibrinogen microcapsules.

Previous studies showed that biomimetic RGD-alginate microcapsules improved the

long-term viability and functionality of encapsulated C2C12 myoblasts, but to the best of

our knowledge this is the first time fibrinogen-alginate have been used for the

encapsulation of MSC(Orive et al. 2009a). Our data indicates that incorporation of

fibrinogen enhances cell viability and proliferation, as well as FIX secretion.

The observed improved proliferation of MSC in fibrinogen-alginate versus non-

supplemented alginate microcapsules is in agreement with the results obtained from our

monolayer cultures (Figure 1). Although further studies are required to analyze the

biology and mechanism of the cell-matrix interactions, fibrinogen may provide matrix

cues that the cells can interact with. It is known that MSCs are able to attach to fibrinogen

through several integrins(Gailit et al. 1997; Schmal et al. 2007; Kisiday et al. 2010).

Therefore, it is speculative, yet conceivable that the enhancement of MSC proliferation


67

and viability observed in this study is modulated by MSCs interacting directly with

fibrinogen. Integrins can directly activate survival pathways via the phosphoinositide 3-

kinase (PI 3-kinase) and mitogen-activated protein kinase (MAPK) pathways and act as

essential cofactors for MSC stimulation by growth factors. Integrins may activate the

MAPK pathway by either of two cascades, one of which involves focal adhesion kinase

(FAK) and the other the adaptor protein Shc (Whitlock et al. 2000a). Karoubi et al.

reported that increased viability of encapsulated MSCs in fibrinogen and fibronectin-

supplemented agarose is likely via the Shc signaling pathway, which is an essential

intermediate of the MAPK cascade(Karoubi et al. 2009b). It has also been demonstrated

that αvβ3 and α5β1, the binding partner integrins of fibrinogen, are among the few

integrins that recruit and activate Shc, and thus play a critical role in the survival of

adherent MSC (Dwayne G Stupack & David A Cheresh 2002; Zvibel et al. 2002).

Improved viability of encapsulated MSCs in fibrinogen-alginate microcapsules

may potentially result from the re-induction of the cell-matrix interaction via the

activation of the extracellular signal-regulated MAPK cascade. Additionally, it was

previously reported that once the MSC reach the local confluence, the rate of proliferation

decreases from the log phase growth(Neuhuber et al. 2008). The acellular regions in non-

supplemented alginate-covered cover slips were likely due to discontinuous and colony-

like proliferation patterns. Conversely, the near homogeneous coverage observed in

fibrinogen-alginate-covered cover slips suggests a better distribution of cells during

proliferation. MSCs were also more homogenous during growth in fibrinogen-alginate

microcapsules compared to non-supplemented microcapsules (data not shown). The


68

increased dispersal of MSCs during proliferation may explain in part their higher viability

and proliferation in fibrinogen-supplemented alginate. Contact inhibition, which slows

cell growth, may be reduced in supplemented microcapsules(Kisiday et al. 2010). The

filopia-like extensions observed by TEM in the cells encapsulated in fibrinogen-alginate

may suggest enhanced attachment. However, such evidence was undetectable by SEM or

confocal microscopy. It is conceivable that testing other fibrinogen concentrations may

lead to different results.

FIX is a clotting factor in the coagulation cascade. Hence, bioavailability of

exogenous FIX and fibrinogen in the microcapsules may promote a thrombogenic state,

thus posing a safety concern. However, and despite the accepted risk for thrombosis in

patients with supraphysiological FIX levels, gene therapy strategies achieving 30-times

the normal FIX level did not cause thrombosis in mice(Ehrhardt & Kay 2002).

Importantly, the concentration of fibrinogen used in the microcapsules (750 g/ml) is

modest when compared to physiological levels of fibrinogen (10 mg/ml). Furthermore,

fibrinogen by itself is unable to lead to fibrin formation without a higher bioavailability of

thrombin, and hemophilic mice implanted with myoblasts secreting similar amounts of

FIX in vitro as MSC did not elicit a thrombotic state, suggesting no additional production

of thrombin. Additionally, UV spectroscopy analysis revealed no detectable leakage of

fibrinogen from microcapsules. Finally, we established the biocompatibility of the

fibrinogen-alginate microcapsules in mice, with no obvious signs of cell overgrowth or

immune response, thus supporting the notion that no fibrinogen leaked from the

microcapsules.


69

We have previously reported the therapeutic secretion of FIX in hemophilic mice

implanted with microcapsules containing G8 myoblasts secreting in vitro ~1500 ng

FIX/106 cells/24 h.(Wen et al. 2006b) Of note, this level is comparable to the FIX

secreted by CB MSCs in this study, thus suggesting the therapeutic potential of

encapsulated MSCs. Future in vivo studies will determine such potential in the treatment

of hemophilia.

Our differentiation experiments demonstrate that encapsulated cells cultured in

osteogenic and basal media exhibit increased viability and proliferation, and consequently

enhanced FIX secretion, compared with encapsulated cells grown in chondrogenic and

adipogenic cultures. Monolayer studies show that FIX transduction of CB MSCs did not

affect the multipotency of cells grown in monolayer culture. Also, CB MSCs differentiate

through mesenchyme lineages only when induced by differentiation media.

Understanding the effect of differentiation induction on encapsulated cell viability and

functionality would allow modulation of cell differentiation status before implantation for

optimum functionality. We showed that high viability, proliferation, and FIX secretion of

encapsulated cells grown in basal and osteogenic media was maintained for at least 28

days. However, cell viability, proliferation and FIX secretion was lower in chondrogenic

and adipogenic cultures. This may suggest the inability of the proposed microcapsule

system to support chondrogenic and adipogenic differentiation. Consistently, it was

reported that cell aggregates are needed to induce chondrogenic differentiation in MSCs

in 3D cultures(A. Goren et al. 2010b). The low cell viability in adipogenic media was

predicted from our monolayer differentiation studies (Figure 5) and was expected due to


70

the inefficiency of CB MSCs to differentiate into the adipogenic lineage(Rebelatto et al.

2008).

5. Conclusion

Encapsulation of recombinant cells continues to hold significant promise for treatment of

diseases caused by protein deficiencies such as hemophilia B. The availability and

plasticity of stem cells make MSCs particularly attractive for future cellular therapies.

However, improvement of viability and functionality of the enclosed cells are still major

challenges of cell encapsulation. Bioactive fibrinogen-alginate microcapsules proposed in

this work enhances cell viability and FIX secretion from the enclosed FIX-engineered

MSCs. Moreover, our results suggest that fibrinogen-alginate provides an appropriate

environment for osteogenic differentiation. Fibrinogen alginate microencapsulation of

FIX-engineered MSCs may have potential for cell-based gene therapy of hemophilia B

and other inherited or acquired protein deficiencies. However, further studies should

address the mechanisms behind improved MSC viability and functionality, as well as the

characterization of the biological interactions of MSCs with the fibrinogen-alginate

matrix.

Acknowledgements

This work was funded in part by a grant from Canadian Blood Services Research &

Development to GH. and AJW, and from NSERC CREATE/IDEM Grant. Thanks to Dr.


71

Frederick A. Ofosu for the generous gift of fibrinogen, and Marcia Reid and Marnie

Timleck for processing the TEM and SEM samples.

AUTHOR DISCLOSURE STATEMENT

The Authors declare that there is no conflict of interest.


72

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75


MESENCHYMAL STEM CELLS IN RGD-ALGINATE VS. FIBRINOGEN-

ALGINATE MICROCAPSULES FOR ENHANCED FIX SECRETION

Authors: Bahareh Sayyar, Megan Dodd, Leah Marquez-Curtis, Anna Janowska-

Wieczorek, Gonzalo Hortelano

Publication Information: Journal of Tissue Engineering and Regenerative Medicine

This paper is ready for submission

Working Hypothesis:

Improved cell viability from the encapsulated MSCs may be obtained by decorating the

core of alginate microcapsules with cross-linked RGD molecules. RGD-alginate and

previously designed fibrinogen-alginate microcapsules may have different effects on FIX

secretion from the encapsulated cells.


76

Encapsulation of FIX-Engineered Human Mesenchymal Stem Cells in RGD-

Alginate vs. Fibrinogen-Alginate Microcapsules for Enhanced FIX Secretion



3, Anna Janowska-Wieczorek

3,4,

Gonzalo Hortelano1,2*

1 School of Biomedical Engineering, McMaster University, Canada

2 Department of Pathology & Molecular Medicine, McMaster University, Canada

3Research & Development, Canadian Blood Services, Canada

4Department of Medicine, University of Alberta, Canada



McMaster University


Ph: +1 (905) 525-9140 ext. 22739

Fax: +1 (905) 521-2613 E-mail: [email protected]



77

Abstract

Cell microencapsulation technology continues to hold significant promise for cell

therapy strategies. The continuous delivery of therapeutic proteins to the host by the

immuno-isolated cells is potentially a convenient and cost-effective strategy to treat a

variety of protein deficiency disorders including hemophilia B. Nevertheless, this

technology has not lived up to expectations mainly due to the compromised viability of

the enclosed cells. Inadequate cell-matrix interactions and subsequent cell death are

among the most critical factors affecting the viability and functionality of the enclosed

cells. In this work, we designed and constructed biomimetic RGD-alginate microcapsules

and evaluated their effect on attachment, viability and FIX secretion of the encapsulated

FIX-engineered human cord blood (CB) mesenchymal stem cells (MSCs). Additionally,

RGD-alginate microcapsules were compared with our previously designed fibrinogen-

microcapsules to compare the effect of peptide (RGD) versus protein (fibrinogen) on cell

viability and functionality. It is shown here that compared with non-modified alginate

matrix, RGD-alginate significantly enhance the viability of the enclosed MSCs. However,

RGD-alginate microcapsules did not significantly affect cell proliferation and FIX

secretion. Like RGD-alginate, fibrinogen-alginate microcapsules significantly improved

the viability of the encapsulated MSC. However, fibronogen-alginate more significantly

increased cell proliferation and subsequent FIX secretion from the encapsulated MSCs

and may be of more practical use in the treatment of hemophilia B or other protein

deficiency diseases.


78

Key words: Hemophilia B, cell microencapsulation, mesenchymal stem cells, alginate,

RGD, fibrinogen


79

1. Introduction

Hemophilia B is an X-linked bleeding disorder caused by human factor IX (FIX)

deficiency which occurs in 1 in 30,000 males (Sadler & Davie 1987; Bolton-Maggs & K.

J. Pasi 2003). Current treatment for severe hemophilia B (patients with <1% active FIX)

involves the life-long and costly infusion of plasma derived or recombinant FIX (Gater et

al. 2011). Gene therapy offers attractive alternative methods to the current treatment. The

most successful gene therapy method to date is the direct injection of viral vectors to

deliver FIX (Nathwani et al. 2011). However, because of the potential safety concerns,

there is a critical need for improving the safety of gene therapy methods (Marshall 2001;

Check 2003).

Encapsulation of recombinant cells is a strategy for continuous delivery of

therapeutic proteins with the potential to treat a wide variety of protein deficiency

diseases such as hemophilia (Orive et al. 2003; Orive et al. 2004; Lim & A. M. Sun 1980;

Koo & T. M. Chang 1993; P. L. Chang et al. 1993; Cieslinski & David Humes 1994;

Wong & T. M. Chang 1986; Aebischer et al. 1994; Aebischer et al. 1986; Orive et al.

2006; Orive et al. 2009; Thakur et al. 2010; G Hortelano et al. 2001; G Hortelano et al.

1996; G Hortelano et al. 1999; Wen et al. 2006; Wen et al. 2007). FIX-engineered cells

can be protected in semi-permeable, biocompatible microcapsules that prevent the

propagation of immune cells while allowing the secretion of transgene proteins. To

overcome the challenge of engineering each individual’s cells, a universal cell line can be

engineered and encapsulated in biocompatible microcapsules to be implanted in different

hemophiliacs.


80

Mesenchymal stem cell (MSC) transplantation is of great interest to be used as a

feasible strategy for the treatment of hemophilia B. MSCs are one of the most promising

types of adult stem cells. They are easy to isolate, can be genetically manipulated to

secrete bioactive molecules, are susceptible to molecules that modify their natural

behavior, and most importantly have an immunosuppressive effect by modulating the

immune function of the cell populations involved in alloantigen recognition and

elimination (Liu & Han 2008).

Biomaterials used for cell encapsulation play a critical role in viability and

functionality of the encapsulated cells. Alginate hydrogels are natural biomaterials used

extensively in cell encapsulation and transplantation because they are biocompatible,

biodegradable and gel rapidly within a solution of divalent ions. While alginates have

many favorable required properties in biomaterials, they are unable to specifically interact

with mammalian cells, which result in decreased cell viability and functionality. One way

to improve performance of alginate biomaterials is to supplement the matrix with

adhesion molecules like native full-length extracellular matrix (ECM) proteins such as

fibronectin, fibrinogen, laminin, or collagen. However, coupling the whole molecules can

lead to nonspecific interactions and is difficult to control. Therefore, a variety of short-

sequence peptides derived from ECM molecules can be readily coupled to alginate and

mediate cell adhesion in place of the larger molecules (Shin et al. 2003).

Arginine glycine aspartic acid (RGD) is an adhesion tri-peptide found in ECM

proteins such as fibronectin, fibrin, and vitronectin (Ruoslahti & Pierschbacher 1986). It

binds directly to several integrin receptors, including V and 5 and is commonly


81

coupled to biomaterials to control adhesion (Boudreau & P. L. Jones 1999; Lee & David J

Mooney 2012). Coupling of RGD peptide to alginate hydrogel has been extensively

studies by Mooney et al. (Jon A Rowley & David J Mooney 2002; Comisar et al. 2007;

Comisar et al. 2006; Bae et al. 2007; J A Rowley et al. 1999). Previous studies reported

that bioactive RGD coupled alginate matrix prolonged the long-term in vitro and in vivo

viability of the seeded cells (Orive et al. 2009; Bidarra et al. 2010; Markusen et al. 2006;

Duggal et al. 2009). We have previously deigned and constructed fibrinogen-alginate

microcapsules for enhanced viability and FIX secretion from the encapsulated MSC. In

this study, we construct RGD-coupled alginate microcapsules and evaluate their effect on

viability and functionality of the encapsulated FIX-engineered MSC.

The main aim and critical challenge of this work is the maintenance and

preservation of long-term viability and functionality of FIX-engineered MSC

encapsulated in alginate-based microcapsules. The objectives of this study are first, to

investigate whether we can manipulate the alginate microcapsules by incorporating RGD

peptide to provide the required cell matrix signaling to increase cell viability; and second,

to study whether the RGD-alginate matrix enhance the FIX secretion from the

encapsulated cells; and third, to compare the effect of RGD-coupled alginate with the

effect of the previously designed fibrinogen-alginate microcapsules on viability,

proliferation and FIX secretion of the encapsulated MSC.


82


2.1. Cell culture

CB samples were obtained after delivery with the mothers’ informed consent in

accordance with the guidelines set out by the University of Alberta Health Research


gradient centrifugation (Amershaw Biosciences, Uppsala, Sweden) and cultured in


supplemented with 10% fetal bovine serum (FBS) and streptomycin (100µg/ml) at 37°C

in 5% CO2. After 24 h, non-adherent cells were removed and complete medium was

replaced. Cells were passaged when they reached about 60% confluency, and cells from

passages 4 to 6 were used in the experiments.

2.2. Engineering of MSC





LentiX 293T cells were transfected with the 4th

generation VSV-G packaging DNA and



MSC cells for 24 h at an MOI of approximately 20. At the time of transduction, CB MSC

were at passage 4 and cultured in 6-well plates at 40% confluency in IMDM.


83

Centrifugation at 1500 rpm for 90 min at 32°C greatly promoted viral transduction

efficiency. Transduced cells were selected using puromycin (3μg/ml) beginning 24 h after

the second round of transduction. Cell media and selection antibiotics were changed

every 2-3 days for 10-14 days.

2.3. Cell encapsulation

MVG ultrapure alginate (MW 291 kDa) and custom-made GRGDSP-coupled

alginate (peptide/alginate molecular ratio of approximately 10/1) made from MVG were

purchased from FMC BioPolymer. Human plasma fibrinogen (Sigma Aldrich, Oakville,

ON, Canada) was incorporated in the MVG alginate solution as described in 2.4 . FIX-

engineered CB MSC were suspended in a RGD-alginate, fibrinogen-alginate and non-

supplemented alginate solution (control) at a concentration of 3x106 cells/ml (1.56%

alginate). Microencapsulation was performed with an encapsulator (Nisco Engineering

Inc., Zurich, Switzerland) as previously described (P. L. Chang et al. 1994). After several

washes, alginate microcapsules were coated with 0.05% (w/v) poly-L-lysine (PLL) and

an outer layer of alginate 0.03% (w/v). All batches were cultured under regular tissue

culture conditions. The same encapsulation procedure was performed on control

experiments.

2.4. Incorporation of fibrinogen into alginate microcapsules

RGD-alginate was purchased from FMC Biopolymer. The GRGDSP was

crosslinked to the MVG alginate with molecular weight of about 280KDa with 7-8


84

peptides per alginate molecules. Fibrinogen was incorporated into the alginate solution at

a concentration of 750 µg/ml of alginate solution. To verify that fibrinogen remained at

the alginate microcapsules following the encapsulation process, samples were taken over

time from the media in which the microcapsules were incubated. Samples were analyzed

using UV spectroscopy (Beckman Coulter TM

, Mississauga, ON, Canada) at 280 nm.

2.5. Assessment of viability and proliferation of encapsulated cells

Viability of encapsulated MSC was assessed using the trypan blue exclusion assay

(Invitrogen, Burlington, Canada) as previously described (Wen et al. 2007; García-Martín

et al. 2002; Thakur et al. 2010). Briefly, a sample of microcapsules was placed on a

microscope slide and crushed by a glass cover slip to release the encapsulated cells.

Subsequently, the cells were stained with trypan blue; dead (blue) and live (transparent)

cells were quantified by inverted light microscope (Leica DM IL).

Proliferation of encapsulated MSC was assessed by MTT assay according to the

manufacturer’s instruction, with modification for encapsulated cells (Thakur et al. 2010).

Briefly, MTT reagent (Sigma-Aldrich) was prepared as a 5 g/ml solution in PBS. 100 μl

of microcapsules were incubated with 10 μl MTT reagent in 96-well plate for 3 hours at

37oC. The encapsulated cells were periodically viewed under an inverted microscope for

presence of intracellular punctate (purple precipitate). After 3 hours, when the purple

precipitate was clearly visible under the microscope, the MTT conversion was stopped

removing the MTT containing medium. The resultant formazan was dissolved in 100 μl

dimethyl sulphoxide (DMSO, BDH Inc. Toronto, Ont., Canada). The plate was incubated


85

in the dark for 3 hours at room temperature. The absorbance of each well was measured at

562 nm in a microtiter plate reader (EL 808, ultra microplate reader, BIO-TEK

Instruments Inc., Winooski, VT, USA).

2.6. F-actin cytoskeleton staining of encapsulated cells

Encapsulated cells (100 μl of capsules) were fixed in 4% paraformaldehyde,

(PFA). Cells were then washed once in pre-warmed PBS and permeabilized by incubation

in 0.1% Triton X-100 in PBS at room temperature for 10 minutes followed by two washes

in PBS. Cells were stained with Alexa Fluor 633 phalloidin (15 μl methanolic stock

solution in 200 μl PBS) (Molecular Probes Invitrogen) for 30 min in the dark. To reduce

nonspecific background staining with these conjugates, 1% bovine serum albumin was

added to the staining solution. A small portion of each stained sample was placed onto a

glass slide and analyzed by inverted confocal microscopy (Zeiss 510, Carl Zeiss Inc.).

2.7. Transmission electron microscopy

All transmission electron microscopy (TEM) studies were done in collaboration

with the electron microscopy facility at the McMaster Children’s Hospital (Hamilton,

ON, Canada).

Alginate microcapsules containing cells were fixed with 2% glutaraldehyde (2%

v/v) in 0.1M sodium cacodylate buffer (pH 7.4). The samples were rinsed 2X in buffer

solution, then post-fixed in 1% osmium tetroxide in 0.1M sodium cacodylate buffer for 1


86

hour. The samples were dehydrated through a graded ethanol (EtOH) series (50%, 70%,

70%, 95%, 95%, 100%, 100%).

The final dehydration for the TEM samples was done in 100% propylene oxide

(PO). Infiltration with Spurr's resin was done through a graded series (2:1 PO:Spurr's, 1:1

PO:Spurr's, 1:2 PO:Spurr's, 100% Spurr's, 100% Spurr's, 100% Spurr's) with rotation of

the samples in between solution changes. The samples were transferred to embedding

moulds which were then filled with fresh 100% Spurr's resin and polymerized overnight

in a 60°C oven. Thin sections were cut on a Leica UCT ultramicrotome and picked

up onto copper grids. The sections were post-stained with uranyl acetate and lead citrate

and then viewed in a JEOL JEM 1200 EX TEMSCAN transmission electron microscope

(JEOL, Peabody, MA, USA) operating at an accelerating voltage of 80kV.

2.8. FIX ELISA assay

Human FIX antigen was detected by ELISA assay, as previously described (Wen

et al. 2006; Wen et al. 2007; Sheffield et al. 2004), using the reagents and protocol from

Affinity Biologicals Inc. (Ancaster, ON, Canada).

2.9. Statistical data analysis


differences existed in the groups of data. Student’s t-test was conducted as a post hoc test

to compare pairs of data. Differences were considered significant for P < 0.05. Data are

expressed as means ± SD.


87

3. Results and Discussion

3.1. Assessment of MSC viability and proliferation in microcapsules

Our study was initiated to study the viability and proliferation of FIX-engineered

MSC in three-dimensional RGD-alginate microcapsules and to evaluate the application of

this cell-based device as a method of FIX secretion for hemophilia B. To characterize the

viability and proliferation of MSC within matrices, encapsulated cells were cultured in

vitro and monitored during 28 days of incubation.

Trypan blue exclusion assay showed high cell viability on day 1 (82% in the

alginate group and 89% in the RGD-alginate group). By day 28, viability dropped to 51%

in alginate microcapsules but remained high (72%) in RGD-alginate microcapsules.

MTT assay viability results also confirm the higher number of viable cells in RGD-

alginate vs. alginate microcapsules (Fig. 1A). The presence of RGD peptides in alginate

gels have been previously shown to enhance the viability of interacting C2C12 cells

(Orive et al. 2009), chondrocytes (Degala et al. 2011), osteoblasts (Evangelista et al.

2007). Also, it was previously observed that GRGDY and GRGDSP coupled alginate

beads substantially retained the viability of encapsulated MSC due to the enhanced cell-

matrix interactions (Markusen et al. 2006; Duggal et al. 2009). Alginate is an inert

biomaterial with no capacity to support cell attachment or interaction due to the lack of

mammalian cell adhesivity and the low protein adsorption to alginate gels (Lee & David J

Mooney 2012; Augst et al. 2006). Integrin receptors on the cell surface have the ability to

interact with the biomimetic cues provided with RGD peptides in RGD-alginate matrix.

Additionally, since the RGD peptides are cross-linked to the alginate matrix, this


88

interaction leads to the attachment of cells to the inner core of the microcapsules while in

suspension. Also, it was previously reported that RGD-alginate matrix can provide an

additional mechanical integrity to the alginate gels via binding interactions between cells

and the adhesion ligands coupled to the alginate chains (Lee & David J Mooney 2012;

Orive et al. 2009; Drury et al. 2005).

MTT assay confirmed that compared with alginate microcapsules, RGD-alginate

microcapsules did not result in statistically significant increase in the proliferation of

encapsulated cells during the 4 week in vitro culture (Fig. 1B). It is shown here that

despite providing adhesion sites using an RGD peptide, encapsulated MSC demonstrate

almost similar proliferation profile whether entrapped in RGD-alginate or non-modified

alginate microcapsules. This is in consistent with previous studies that report the inability

of MSC to proliferate within either 1% or 1.8% RGD-alginate microcapsules (Duggal et

al. 2009). It is possible that other concentrations of alginate or different hydrogel/peptide

properties may result in increased proliferation of the encapsulated MSC.


89

Figure 1. Viability and proliferation of human cord blood-derived mesenchymal stem cells (CB MSCs). (A)

The effect of RGD-alginate on viability of encapsulated cells. The ratio of viable cells in RGD-alginate to

the viable cells in alginate was calculated using the MTT assay. (B) Comparison of MSC proliferation in

RGD-alginate and alginate (no significant difference). The ratio of viable cells per viable cells at day 1 was

calculated using the MTT assay. Data are means ±SD, n=3, Student’s t-test. *Significant difference from

1.0, P<0.05.


90

3.2. FIX secretion from encapsulated cells

Aside from studying the effect of RGD-alginate microcapsules on viability and

proliferation of encapsulated FIX-MSC, FIX ELISA assay was used to evaluate their

effect on FIX secretion. Fig. 2 compares the FIX secretion profile of MSC encapsulated

in RGD-alginate and non-modified alginate microcapsules during the 28 days of in vitro

culture. Incorporation of RGD molecules in the alginate matrix resulted in slightly

enhanced FIX secretion from the encapsulated MSC on most of the days during the

experiment. However, this increase in FIX secretion is not statistically significant. It is

shown here that the enhanced viability of encapsulated cells does not result in statistically

significant increased FIX secretion. We have previously reported significantly enhanced

FIX secretion from MSC encapsulated in fibrinogen-supplemented alginate

microcapsules. To further study the factors that affect the FIX secretion, the RGD-

coupled and fibrinogen-supplemented microcapsules are compared in the following

section.


91

Figure 2. FIX secretion from human cord blood-derived mesenchymal stem cells (MSC). Comparison of

FIX secretion from the MSC in alginate and RGD-alginate. FIX secretion was measured using the ELISA

and reported as the mass of FIX (ng) secreted from 1 ml of microcapsules per 24 hr. Data are means ±SD,

n≥3. P<0.01, Student’s t-test. No significant difference RGD-alginate vs. alginate on each day.

3.3. Effect of RGD-alginate vs. fibrinogen-alginate on cell viability and FIX secretion

As shown in Figure 3A, both RGD-alginate and fibrinogen-alginate microcapsules

significantly enhance the viability of the encapsulated cells during 28 days of in vitro

growth. Cell death resulting from loss of interaction with environment in adherence-

dependent cells, involves a series of complex cascades that depend on the cell type and

the type of cell-matrix interactions. Cell-matrix interactions in three dimensions are very

complex and are poorly defined for MSC. We have previously explained several reasons

for enhanced viability of cells encapsulated in fibrinogen-alginate microcapsules. This

improved viability potentially results from the re-induction of the cell-matrix interaction,

via the activation of the extracellular signal-regulated MAPK cascade. Karoubi et al.

reported that increased viability of encapsulated MSC in adhesion protein-supplemented

agarose is likely via Shc signaling pathway that is an essential intermediate of the MAPK

cascade (Karoubi et al. 2009). It has also been demonstrated that αvβ3 and α5β1, the

binding partner integrins of fibrinogen, are among the few integrins that recruit and

activate Shc (X. Q. Wang et al. 2001) and play a critical role in the survival of adherent

MSC (Karoubi et al. 2009). In cell-adhesive proteins such as fibrinogen and fibronectin,

RGD is the recognition sequence that is recognized and bound by the αvβ3 and α5β1

integrins (Barczyk et al. 2010) and therefore plays a crucial role in cell attachment.


92

Figure 3B compares the effect of RGD-alginate and fibrinogen-alginate

microcapsules on FIX secretion from the encapsulated MSC. Here, FIX secreted (ng

FIX/ml capsules/ml media/24 hr) from RGD-alginate or fibrinogen-alginate

microcapsules is normalized to the secretion value from non-supplemented alginate

microcapsules. As shown in this figure, incorporation of fibrinogen within alginate

microcapsules results in significantly enhanced FIX secretion from the encapsulated cells,

which is not detected in cells encapsulated in RGD-alginate microcapsules.

Our previous study demonstrates that fibrinogen-alginate microcapsules

significantly increased the proliferation of the encapsulated MSC. In this study, the MTT

assay confirmed that incorporation of RGD peptide into the alginate microcapsules did

not result in significantly increased proliferation of encapsulated cells (Fig. 2B).

Considering that both RGD-alginate and fibrinogen-alginate microcapsules improved the

viability of the encapsulated cells to the same degree, it is likely that compared to the

improved viability of the cells, enhanced proliferation has a more significant effect in

improving the FIX secretion from the encapsulated cells.


93

Figure 3. Effect of RGD or fibrinogen modification on viability and FIX secretion of encapsulated cells. (A)

Effect of RGD- vs. fibrinogen-modification on viability of the encapsulated MSC. (B) Effect of RGD- vs.

fibrinogen-modification on FIX secretion from the encapsulated MSCs. *Significant difference from 1.0,

P<0.05. **Significant difference from 1.0, P<0.09.

3.4. Cell-matrix analysis

For an in-depth analysis of cell-matrix interactions within microcapsules, we

examined the encapsulated cells within RGD-alginate, fibrinogen-alginate and non-


94

supplemented alginate using confocal microscopy and transmission electron microscopy

(TEM) (Fig. 4).

Confocal analysis demonstrates that cells encapsulated in non-supplemented

alginate were completely rounded with strong F-actin filament expression close to the cell

membrane (Fig. 4C). Additionally, there is not a significant difference between the cells

encapsulated in fibrinogen-alginate and non-supplemented alginate microcapsules in

terms of morphology or F-ac tin staining. There were no distinct patterns or alignment of

the filaments in these cells (Fig. 4B,C). However, it is shown that cells encapsulated in

RGD-alginate microcapsules acquired a compact shape but depicted a more intricate

pattern of F-actin filaments with more defined structure and distinct pattern (Fig. 4A).

Similar results were seen in a previous study of MSC in 1.8% RGD-alginate beads where

the cells acquired a compact shape (Duggal et al. 2009). However, these results differ

from those described in another study of MSC in 1% RGD alginate where the cells

demonstrated an elongated shape after a few days in alginate. Most likely, the observed

differences are due to the different concentrations of alginate or different RGD/alginate

ratio used in these studies.

As detected by the TEM, cells encapsulated in RGD-alginate microcapsules

clearly demonstrate the presence of filopia-like membrane extensions into the

surrounding matrix (Fig. 4D). Membrane extensions are less clearly demonstrated by the

cells encapsulated in fibrinogen-alginate matrix (Fig. 4E) and are not observed in cells

encapsulated in non-supplemented alginate microcapsules (Fig. 4F). As expected,

considering that RGD molecules were cross-linked to the alginate matrix, cell attachment


95

is more clearly observed in RGD-alginate microcapsules. Cell attachment to the non-

cross-linked fibrinogen-supplemented alginate matrix is less noticeably observed. It is

also possible that fibrinogen concentrations other than tested here could affect cell

attachment and lead to different results.

Figure 4. (A-C) F-actin staining of encapsulated cells. (D-F) TEM images of encapsulated cells

4. Conclusions

Cell encapsulation is one of the most important strategies for cell-based therapies.

Yet, these cell-based systems are not being clinically used due largely to their high

immunogenic potentials and compromised viability and functionality of the encapsulated

cells. This work involved design and construction of RGD-alginate microcapsules to


96

encapsulate CB MSC for FIX delivery. Compared with the non-supplemented alginate

microcapsules, RGD-alginate microcapsules significantly improved the viability of the

encapsulated cells. The key advantage of RGD-alginate microcapsules is that while the

hydrogel matrix encapsulates the cells and protects them from the host immune response,

the RGD molecules cross-linked to the alginate matrix at the core of the microcapsule

enhance cell viability by providing the cells with attachment site while in suspension.

However, considering the insignificant effect of RGD-alginate matrix on cell

proliferation, FIX secretion was not significantly improved from RGD-alginate

microcapsules. As RGD-alginate, fibrinogen-supplemented alginate microcapsules

significantly enhanced the viability of encapsulated MSC but more significantly increased

the cell proliferation and subsequent FIX secretion from the encapsulated MSC. Viability

and FIX secretion data of encapsulated cells suggest that although both RGD-alginate and

fibrinogen-alginate microcapsules provide biomimetic 3-D environment for encapsulated

MSC applicable in cell therapy technologies, fibrinogen-alginate microcapsules more

significantly enhance the proliferation and protein secretion from the encapsulated cells

may have potential for cell-based gene therapy of hemophilia B and other inherited or

acquired protein deficiencies.

Acknowledgements

This work was funded in part by a grant from Canadian Blood Services to G. H. and

A.J.W. Thanks to Dr. Frederick A. Ofosu for the generous gift of fibrinogen, and Marcia

Reid and Marnie Timleck for processing the TEM and SEM samples.


97

Declaration of Interest



98

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102

CHAPTER 5: FIBRONECTIN-ALGINATE MICROCAPSULES IMPROVE CELL

VIABILITY AND PROTEIN SECRETION OF ENCAPSULATED FIX-

ENGINEERED HUMAN MESENCHYMAL STEM CELLS

Authors: Bahareh Sayyar, Megan Dodd, Leah Marquez-Curtis, Anna Janowska-

Wieczorek, Gonzalo Hortelano

Publication Information: Journal of Tissue Engineering Part A

This paper is ready for submission

Working Hypothesis:

Improved cell viability and FIX secretion from the encapsulated MSCs may be obtained

by decorating the core of alginate microcapsules with fibronectin molecules.

Concentration of fibronectin in alginate matrix may affect the viability, proliferation, and

FIX secretion from the encapsulated cells. Moreover, differentiation induction of the

encapsulated cells through osteogenic, chondrogenic, or adipogenic media may affect the

viability and functionality of the encapsulated cells.


103

Fibronectin-Alginate Microcapsules Improve Cell Viability and Protein Secretion of

Encapsulated FIX-Engineered Human Mesenchymal Stem Cells



2, Anna janowska-Wieczorek

2,3,

Gonzalo Hortelano1

1School of Biomedical Engineering, McMaster University, Hamilton, Ontario, Canada

2 Research & Development, Canadian Blood Services, Edmonton, Alberta, Canada

3 Department of Medicine, University of Alberta, Edmonton, Alberta, Canada



McMaster University


Ph: +1 (905) 525-9140 ext. 22739

Fax: +1 (905) 521-2613 E-mail: [email protected]



104

Abstract

Continuous delivery of proteins by engineered cells encapsulated in biocompatible

polymeric microcapsules is of considerable therapeutic importance. However, this

technology has not lived up to expectations. Inadequate cell-matrix interactions and

subsequent cell death are among the critical factors that limit the application of

recombinant cell microencapsulation for hemophilia treatment. In this work we

hypothesize that the use of fibronectin-alginate matrix may result in enhanced viability

and functionality of encapsulated FIX-engineered cord blood-derived human

mesenchymal stem cells (MSCs) in suspension culture. MSCs were encapsulated in

alginate-based microcapsules containing 10, 100, or 500µg/ml fibronectin to ameliorate

cell survival. MSCs within 100 and 500µg/ml fibronectin microcapsules demonstrate

improved cell viability and proliferation and greater FIX secretion compared to the cells

within non-supplemented microcapsules. 10µg/ml fibronectin microcapsules did not

significantly affect viability and protein secretion from the encapsulated cells.

Differentiation studies demonstrate osteogenic, not chondrogenic or adipogenic,

differentiation capability and efficient FIX secretion of the enclosed MSCs in the

fibrinogen-alginate suspension culture. Thus, the use of recombinant MSCs encapsulated

in fibronectin-alginate microcapsules in basal or osteogenic cultures may be of practical

use in the treatment of hemophilia B or other protein deficiency diseases.

KEYWORDS

Hemophilia B, cell encapsulation, alginate, fibronectin


105

Introduction

Hemophilia B is an X-linked bleeding disorder caused by lacking of functional

circulating FIX (Sadler & Davie 1987; Bolton-Maggs & K. J. Pasi 2003). Current

treatment for severe hemophilia B patients (patients with <1% active FIX) involves the

costly and long-life injection of plasma-derived or recombinant FIX. Gene therapy offers

an attractive alternative treatment to hemophilia B. Current successful hemophilia B gene

therapy strategies involve direct injection of viral vectors for FIX delivery but there are

some associated complications regarding the host immune response (Nathwani et al.

2011; Marshall 2001; Check 2003). Cell therapy technologies offer a variety of safety

advantages for hemophilia treatment. Encapsulation of recombinant cells within a semi-

permeable membrane is a strategy for continuous delivery of therapeutic proteins for a

variety of protein deficient diseases such as hemophilia B (Orive et al. 2003; Orive et al.

2004; Lim & Sun 1980; Koo & T. M. Chang 1993; P. L. Chang et al. 1994a; Cieslinski &

David Humes 1994; Orive et al. 2009; G Hortelano et al. 2001; G Hortelano et al. 1996;

G Hortelano et al. 1999; Wen et al. 2007; Wen et al. 2006; Thakur et al. 2010). Current

cell therapy strategies for hemophilia treatment are limited mainly due to the

compromised viability of the implanted recombinant cells.

It is well known that adhesive peptides or proteins play central roles at the cell-

surface interface through interacting with integrins for mediating survival signals (Shin et

al. 2003). Several studies have addressed the significance of selected proteins such as

laminin, fibrinogen, and fibronectin to enhance cell survival and have reported different

results depending on the cell line and cell-biomaterial combination (Karoubi et al. 2009;


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Stupack & Cheresh 2002; Whitlock et al. 2000). We have previously designed and

constructed fibrinogen-alginate and RGD-alginate microcapsules for enhanced viability

and functionality of the enclosed FIX-engineered mesenchymal stem cells (reference the

thesis). Here, we explore the effect of fibronectin as a cell-adhesive molecule on viability

and protein secretion of encapsulated MSCs within alginate-based microcapsules.

Fibronectin, one of the most important ECM proteins, is a multidomain

glycoprotein possessing binding sites for a variety of other ECM components including

collagen, heparin A and B, fibrin, and chondroitin sulfate. It is present both in blood (150-

300 g/ml) and in the ECM and is of critical importance for linking cells to the ECM via

the cell surface integrins and adaptor molecules (Schoen Fredrick J. & Mitchell Richard

N. n.d.; Dolatshahi-Pirouz et al. 2011).

The objectives of this study are: First, to assess if the incorporation of fibronectin

within alginate matrix can manipulate the alginate microcapsules for enhanced viability

and FIX secretion level of umbilical cord blood-derived (CB) MSC. Second, to

investigate how the different concentrations of fibronectin can affect cell viability,

proliferation and FIX secretion. Third, to induce differentiation of the encapsulated CB

MSC in fibronectin-alginate microcapsules into osteogenic, chondrogenic and adipogenic

lineages in order to examine the effect of differentiation status of the encapsulated cells

on cell viability and FIX secretion.


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2.1 Cell Culture

Umbilical cord blood was obtained after delivery with the mothers’ informed

consent in accordance with the guidelines of the University of Alberta Health Research


gradient centrifugation (Amersham Biosciences, Uppsala, Sweden) and cultured in


supplemented with 10% fetal bovine serum and streptomycin (100 µg/ml) at 37°C in 5%

CO2. After 24h, non-adherent cells were removed and complete medium was replaced, as

described previously (Son et al. 2006). MSCs were used in experiments before reaching

passage 6. Flow cytometric analysis showed positive expression for CD90 and CD105 but

not for the hematopoietic markers CD34 and CD 45 (data not shown).

2.2 Engineering of MSC





LentiX 293T cells were transfected with the 4th

generation VSV-G packaging DNA and




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MSCs (passage 4) for 24 h at a MOI of approximately 20. Transduced cells were selected

with puromycin (3 μg/ml) (Clontech) after 15 day incubation.

2.3 Cell encapsulation

MVG ultrapure alginate was purchased from FMC, BioPolymer (Philadelphia,

PA, USA). FIX-engineered CB MSC were suspended in a fibronectin-alginate and non-

supplemented (control) alginate solution (1.56% alginate) at a concentration of 3x106

cells/ml. Microencapsulation was performed with an electrostatic encapsulator (Nisco

Engineering Inc., Zurich, Switzerland) as previously described (P. L. Chang et al. 1994b).

Briefly, cell suspension was pumped through the electrostatic encapsulator (voltage: 7kV)

at the flow rate of 0.9 ml/min into a vial containing 1.1% CaCl2 yielding microcapsules

400 μm in diameter. Cell-loaded beads were then washed with saline solution and cross-

linked with poly-L-lysine and with an outer layer of non-supplemented alginate. Human

plasma fibronectin (Sigma Aldrich, Oakville, ON, Canada) was added to the alginate core

as necessary at concentrations of 10, 100, and 500 µg/ml of cell-alginate solution.

2.4 Differentiation of human MSCs

For induction of osteogenic, chondrogenic, or adipogenic differentiation,

encapsulated MSC were cultured in StemPro Osteogenic, StemPro Chondrogenic, or

StemPro Adipogenic differentiation medium (GIBCO Invitrogen, Burlington, ON,

Canada), respectively and with appropriate supplements.


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At week 3-post osteogenic induction and at week 2-post chondrogenic and

adipogenic induction, encapsulated cells were washed with phosphate buffered saline

(PBS), fixed in 4% paraformaldehyde for 10 min and washed. Encapsulated cells were

stained with Alizarin Red, Alcian Blue, and Oil Red O dyes (Sigma Aldrich) for detection

of calcium deposits, proteoglycans, and fat vacuoles as an indication of osteogenic,

chondrogenic, and adipogenic differentiation, respectively. Microcapsules were

visualized under an inverted light microscope (Leica DM IL, Leica Microsystems,

Richmond Hill, ON, Canada).

2.5 Assessment of viability and proliferation of encapsulated cells

Viability and proliferation of encapsulated MSC was assessed using the

Live/Dead viability assay (Sigma Aldrich, Oakville, ON, Canada) according to the

manufacturer’s instruction adapted to encapsulated cells.

2.6 F-actin cytoskeleton staining of encapsulated cells

Encapsulated cells (100 μl of capsules) were fixed in 4% paraformaldehyde. As

indicated in manufacturer’s protocol, cells were then washed in pre-warmed PBS and

permeabilized by incubation in 0.1% Triton X-100 in PBS for 10 minutes, followed by

two washes in PBS. Cells were stained with Alexa Fluor 633 phalloidin (15 μl methanolic

stock solution in 200 μl PBS, for 30 min in the dark) (Molecular Probes Invitrogen,

Burlington, ON, Canada) containing 1% bovine serum albumin to reduce nonspecific


110

background. A sample was analyzed by inverted confocal microscopy (Zeiss 510, Carl

Zeiss Inc., Toronto, Ontario, Canada).

2.7 Transmission electron microscopy

Transmission electron microscopy (TEM) studies were done at the electron

microscopy facility, McMaster Children’s Hospital (Hamilton, ON, Canada).

Encapsulated cells were fixed with 2% glutaraldehyde (2% v/v) in 0.1M sodium

cacodylate buffer (pH 7.4). The samples were rinsed 2X in buffer solution, then post-

fixed in 1% osmium tetroxide in 0.1M sodium cacodylate buffer for 1 hour and

dehydrated through a graded ethanol (EtOH) series (50%, to 100%). The final

dehydration of the TEM samples was done in 100% propylene oxide (PO). Infiltration

with Spurr's resin was done through a graded series of PO:Spurr’s 2:1, 1:1, 1:2, 0:1 with

rotation of the samples in between solution changes. The samples were transferred to

embedding moulds which were then filled with fresh 100% Spurr's resin and polymerized

overnight in a 60°C oven. Thin sections were cut on a Leica UCT ultramicrotome (Leica

Microsystems, Richmond Hill, ON, Canada) and picked up onto copper grids. Sections

were post-stained with uranyl acetate and lead citrate and viewed in a JEOL JEM 1200

EX TEMSCAN transmission electron microscope (JEOL, Peabody, MA, USA) at an

accelerating voltage of 80kV.


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2.8 FIX ELISA assay

Human FIX antigen in culture media was quantified by an ELISA assay (Affinity

Biologicals Inc., Ancaster, ON, Canada), as previously described (Wen et al. 2006).

2.9 Statistical data analysis


differences existed in the groups of data. Student’s t-test or Tukey HSD was conducted as

post hoc tests to compare the pairs of data. Differences were considered significant when

p<0.05. Data are expressed as means ± SD.

3. Results

3.1 Assessment of cell viability and proliferation

We applied the LIVE/DEAD cell viability assay as a primary method for cell

viability and proliferation analysis. Encapsulated MSCs were monitored during one

month in vitro culture. To enhance the survival conditions within the microcapsules,

alginate matrix was supplemented with different concentrations of human plasma

fibronectin molecules, 10, 100, or 500 g fibronectin/ml cell-alginate mixture. No

significant difference was observed in the %viability of encapsulated cells on day 1,

77±4% in control, 79±3% in 10g/ml fibronectin-alginate (10 Fibronectin), 77±4% in

100g/ml fibronectin-alginate (100 Fibronectin), and 78±3% in 500g/ml fibronectin-

alginate (500 Fibronectin). After two weeks of in vitro culture, %viability was

significantly higher for the cells enclosed within 100 Fibronectin and 500 Fibronectin


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compared to the cells within control microcapsules. However, compared to control, 10

Fibronectin did not affect the cell viability significantly. On day 28, %viability in control

and 10 Fibronectin microcapsules dropped to 54±2%, and 61±2%, respectively. However,

it remained high for cells within 100 Fibronectin, 67±2%, and 500 Fibronectin, 76±7%,

(Figure 1,A).

To better compare 10, 100, and 500 Fibronectin in terms of their effect on the cell

viability, total number of viable cells in each fibronectin-supplemented group were

normalized to total number of viable cells in the control group (Figure 1,B). It is evident

that after two weeks of culture, 100 Fibronectin and 500 Fibronectin groups, but not 10

Fibronectins group, significantly improved the number of viable cells compared with the

control group.


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Figure 1. %Viability of CB MSCs. The effect of incorporation of 10, 100, and 500 mg/ml fibronectin on

(A) %viability of the encapsulated cells, (B) viable cells (%control). Data are means ± SD, n=4, Tukey

HSD, , *Significant difference of 100 Fibronectin and 500 Fibronectin from control, **Significant

difference of 100 Fibronectin and 500 Fibronectin from 10 Fibronectin, P<0.05.

Additionally, incorporation of 100g/ml and 500g/ml fibronectin into the

alginate microcapsules resulted in enhanced proliferation of the encapsulated MSCs


114

during the in vitro growth. After two weeks of in vitro culture, number of viable cells

(%number of viable cells on day 1) was comparable in 100 Fibronectin and 500

Fibronectin and was significantly higher than that of the cells in the control (Figure 2,A).

After one week of in vitro culture, both 100 Fibronectin and 500 Fibronectin significantly

increased the total number of cells, which reached to approximately 130% of the control

on day 28. On the other hand, compared with the control group, the 10 Fibronectin did

not affect the total number of cells significantly (Figure 2,B).


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Figure 2. Proliferation of CB MSCs. (A) The effect of fibronectin-alginate microcapsules on viability of

encapsulated cells. (B) Comparison of total number of cells in fibronectin-alginate microcapsules. Cell

proliferation was calculated using the LIVE/DEAD viability assay. Data are means ± SD, n=4, Tukey

HSD, . *Significant difference of 100 Fibronectin and 500 Fibronectin from control, **Significant

difference of 100 Fibronectin and 500 Fibronectin from 10 Fibronectin, P<0.05.


116

3.2 FIX secretion from encapsulated cells

Consistent with improved viability and proliferation, incorporation of 100g/ml

and 500g/ml fibronectin within the alginate matrix resulted in enhanced FIX secretion

from the encapsulated cells. As measured by ELISA, FIX secretion from 100 Fibronectin

and 500 Fibronectin was above 3000 ng/ml (capsules)/24 hr on day 14 and remained

above 2000 ng/ml (capsules)/24 hr after 28 days of in vitro culture. On the other hand,

FIX secretion from 10 Fibronectin and control groups was approximately 2000 ng/ml

(capsules)/24 hr on day 14 and decreased to around 1000 ng/ml (capsules)/24 hr on day

28 (Figure 3,A).

To better compare the FIX secretion profile from different microcapsules, FIX

secreted from fibronectin-alginate microcapsules were normalized in respect to the FIX

secretion value from the control microcapsules (Figure 3,B). It is shown here that after

two weeks of in vitro culture, FIX secretion from 100 Fibronectin and 500 Fibronectin,

but not 10 Fibronectin, was significantly higher than FIX secretion from the control. On

day 28, FIX secretion from 100 and 500 Fibronecin reached to around 210% of FIX

secretion from the control and 10 Fibronectin.


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Figure 3. FIX secretion by CB MSC. Comparison of FIX secretion from MSCs in non-supplemented

(control), fibronectin-supplemented microcapsules (10, 100, 500 fibronectin). FIX secretion was measured

using ELISA assay and reported as (A) the amount of FIX (ng) secreted from 1 ml of microcapsules in 24

h. (B) the amount of FIX secretion from fibronectin-alginate microcapsules/ the amount of FIX secretion

from control microcapsules. Data are means ± SD, n=4. Tukey HSD, . *Significant difference of 100


118

Fibronectin and 500 Fibronectin from control, **Significant difference of 100 Fibronectin and 500

Fibronectin from 10 Fibronectin, P<0.05.

It was of interest to detect whether or not the enhanced FIX secretion profile in

100 and 500 Fibronectin was only due to the increased number of viable cells in these two

microcapsule systems. For this, we introduced a parameter called the FIX secretion index

(ng FLX secreted from 106 viable cells/24 hr) and compared its value among 10

Fibronectin, 100 Fibronectin, 500 Fibronectin, and control microcapsules during one

month in vitro culture. As shown in Figure 4, on day 28, FIX secretion index in 100 and

500 Fibronectin is significantly higher than that of the control. This suggests the positive

effects of these two microcapsule systems on the protein secretion capability of the

enclosed cells beside their effect on improved viability and proliferation.


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Figure 4. FIX secretion index (ng FLX secreted from 106 viable cells/24 hr) of fibronectin-alginate and

non-supplemenetd alginate microcapsules during one-month in vitro culture. Data are means ± SD, n=4.

Tukey HSD, . *Significant difference from control.

3.3 Cell-matrix analysis

For an in-depth look at the cell-matrix interactions, encapsulated MSCs were

examined using transmission electron microscopy (TEM; Figure 5). Cells encapsulated in

both 100 Fibronectin and 500 Fibronectin demonstrated the presence of filopia-like

membrane extensions into the surrounding matrix (Figure 5, E-H), which were observed

to a less extent in 10 Fibronectin (Figure 5, C-D) and was not observed in the control

group (Figure 5, A-B). However, cell-cytoskeleton studies using confocal microscopy

showed that encapsulated cells in non-supplemented and all fibronectin-supplemented

microcapsules were rounded with no distinct alignment of F-actin filaments (data not

shown).


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Figure 5. Transmission electron microscopy images of CB MSCs encapsulated in control microcapsules

(A,B); 10 Fibronectin microcapsules (C,D); 100 Fibronectin microcapsules (E,F); 500 Fibronectin

microcapsules (G,H). Cells were fixed 10 days after encapsulation and processed for electron microscopy.

Bars indicate 2 microns

3.4 Effect of differentiation induction on cell viability and FIX secretion

It is well known that MSCs have the capacity to differentiate into osteogenic,

chondrogeniic, and adipogenic lineages. Understanding the effect of differentiation

induction on cell viability and functionality would allow modulation of cell

differentiation status before implantation for optimum functionality. Therefore in this

study, the effect of the differentiation induction on cell viability, proliferation, and FIX

secretion was assessed.

As shown before, 100 Fibronectin and 500 Fibronectin microcapsules were not

significantly different from each other in terms of their effects on improving the


121

encapsulated cell viability and functionality. Hence, we chose the microcapsules with

lower fibronectin concentration (100 Fibronectin) as a model for differentiation analysis.

MSCs in 100 Fibronectin microcapsules were cultured in osteogenic,

chondrogenic, and adipogenic medium. Some capsules were grown in control basal

medium. LIVE/DEAD viability assay was used to measure the number of viable cells in

each group. The total number of viable cells grown in differentiation media was

normalized with respect to the total number of viable cells in the basal medium (Figure

6,A). It is shown here that the viability of MSCs cultured in osteogenic medium is not

significantly different from that of the cells grown in basal medium and is significantly

higher than the viability of the cells grown in chondrogenic or adipogenic media. The

lower viability of cells cultured in chondrogenic and adipogenic media suggests the

inability of the 100 Fibronectin alginate microcapsules to effectively support

chondrogenic and adipogenic differentiation. This inability is further confirmed by cell

staining protocols.

The effect of differentiation induction on FIX secretion from encapsulated cells

was also investigated. The low FIX secretion from the encapsulated cells grown in

chondrogenic and adipogenic media is consistent with the lower number of viable cells in

these conditions compared with the number of viable cells in basal or osteogenic media.

Consistent with the cell viability, FIX secretion from the cells grown in osteogenic

medium is not significantly different from the FIX secretion value from the cells cultured

in basal medium (Figure 6,B). Also, comparing the FIX secretion index reveals that

encapsulated cells grown in basal and osteogenic medium have the same FIX index


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values and therefore are comparable to each other in terms of their ability for FIX

secretion.

Figure 6. Viability and FIX secretion of CB MSCs in differentiation media. (A) Number of viable MSC in

100 Fibronectin microcapsules cultured in differentiation media normalized to the number of viable cells

grown in basal media. (B) Comparison of FIX secretion from the MSCs in 100 Fibronectin microcapsules

grown in differentiation media. Data are means ± SD, n 3. Viable cells were measured calculated using


123

the LIVE/DEAD viability assay. FIX secretion was measured using ELISA. p<0.05, Student’s t-test.

*Significant difference from 100%.

MSCs grown in 100 Fibronectin-alginate suspension culture were also evaluated

for their ability to differentiate into the 3 mesoderm lineages, osteoblasts, chondrocytes,

and adipocytes. To study the differentiation status of the cells, encapsulated MSCs were

stained by appropriate stains to detect osteogenic, chondrogenic, and adipogenic

differentiation (Figure 7). As shown, even with no stain, calcium deposit is evident in the

microcapsules grown in osteogenic culture (Figure 7,B). Presence of calcium deposits

were further confirmed by Alizarin red, 3 weeks post encapsulation (Figure 7,D). The

encapsulated cells grown in chondrogenic or adipogenic media were not successfully

differentiated into chondorcytes or adipocytes (Alcian Blue, Oil Red-O, 2 weeks post

encapsulation). (Figure 7,E-H).

Figure 7. Differentiation potential of microencapsulated FIX-engineered CB MSCs. (A-D) Osteogenic

differentiation analysis, (A) unstained in basal medium, (B) unstained in osteogenic medium, stained with

Alizarin Red in basal (C) and osteogenic medium (D); (E-F) Adipogenic differentiation analysis, stained


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with Oil Red-O in basal (E) and adipogenic medium (F); (G-H) Chondrogenic differentiation analysis,

stained with Alcian Blue in basal (G) and chondrogenic medium (H).

4. Discussion

In this study we investigate FIX-engineered human cord blood-derived

mesenchymal stem cells in biomimetic fibronectin-alginate microcapsules. We further

evaluate the effect of different concentrations of supplemented fibronectin on behavior of

the encapsulated cells. Our data indicates that incorporation of 100g/ml and 500g/ml

fibronectin, but not 10g/ml fibronectin, within the alginate microcapsules reduced cell

death and significantly enhanced cell viability and proliferation compared to the non-

supplemented control group. Karoubi et al. have previously reported enhanced viability

and greater metabolic activity of human marrow stromal cells encapsulated in agarose

microcapsules incorporated with fibrinogen and fibronectin molecules due to re-

introduction of cell-matrix interactions (Karoubi et al. 2009). In our previous studies, we

have reported enhanced viability of MSCs encapsulated in RGD-alginate or fibrinogen-

alginate microcapsules (reference the thesis). Improved proliferation was also detected

for the cells encapsulated within protein-supplemented alginate (fibronogen-alginate) but

not for the cells within RGD-coupled alginate (RGD-alginate). Although further studies

are required to analyze the mechanisms of cell-matrix interactions, it can be discussed

that as a cell adhesion molecule, fibronectin has the advantage of providing the cells with

matrix cues for enhanced interaction and subsequent survival and proliferation. MSCs are

able to attach to fibronectin through several integrins, which may activate the suvival


125

pathways phosphoinositide 3-kinase (PI 3-kinase) and mitogen-activated protein kinase

(MAPK). Integrins can activate the MAPK pathway by either of two cascades, one of

which involves focal adhesion kinase (FAK) and the other the adaptor protein Shc

(Whitlock et al. 2000). It has been previously demonstrated that integrins αvβ3 and α5β1,

the binding partner integrins of fibronectin, are among the few integrins that recruit and

activate Shc and play a critical role in the survival of adherent MSC (Stupack & Cheresh

2002; Zvibel et al. 2002). Karoubi et al. reported that increased viability of encapsulated

MSCs in fibrinogen and fibronectin-supplemented agarose is likely via the Shc signaling

pathway, an essential intermediate of the MAPK cascade (Karoubi et al. 2009).

Therefore, it is speculative, yet conceivable that the enhancement of MSC proliferation

and viability observed in this study is modulated by MSCs interacting directly with

fibronectin.

It is widely accepted that cell shape reveals critical information about the adherent

cells (Kilian et al. 2010; McBeath et al. 2004; Dolatshahi-Pirouz et al. 2011). A non-

flatted and round shape is typical for non-proliferating cells which are likely to enter the

apoptosis pathways while a well-spread cell shape is an indicator of a healthy cell

(Dolatshahi-Pirouz et al. 2011). Based on the images on Figure 4, we postulate that the

presence of 100 and 500 g/ml fibronectin molecules within the alginate microcapsules is

directing formation of filopia-like membrane extensions in to the matrix that

demonstrated cell attachment. These membrane extensions are significantly less evident

for the cells encapsulated in 10 Fibronectin and not detectable for cells in control group.

Fibronectin molecules potentially act to re-introduce the necessary cell-matrix


126

interactions required for cell survival and proliferation. It is possible that other

concentrations of alginate or chemical cross-linking the fibronectin molecules to the

alginate matrix may result in stronger cell-matrix attachment that can be detectable by

confocal analysis.

Encapsulation of FIX-engineered MSC in 100 and 500 Fibronectin microcapsules

resulted in enhanced FIX secretion, which is not detected in cells cultured in 10

Fibronectin or control microcapsules. It is worthy of note that the enhanced FIX secretion

from the 100 Fibronectin and 500 Fibronectin microcapsules is not only due to the

increased number of viable cells in these two microcapsules. Figure 4 demonstrates that

on day 28, FIX secretion index (ng FLX secreted from 106 viable cells/24 hr) in 100 and

500 Fibronectin, but not 10 Fibronectin, is significantly higher than that of the control.

Therefore, 100 and 500 Fibronectin microcapsules enhance the protein secretion ability of

the enclosed cells beside their viability and proliferation.

Understanding the effect of differentiation induction on encapsulated cell behavior

would allow modulation of cell differentiation status before implantation for optimum

functionality. Our data suggest that encapsulated cells within 100 Fibronectin

microcapsules cultured in osteogenic and basal media exhibit enhanced viability,

proliferation, and FIX secretion compared with the encapsulated cells grown in

chondrogenic and adipogenic media for at least 28 days. Also, comparing the FIX

secretion index reveals that encapsulated cells grown in basal and osteogenic medium

have the same FIX index values and therefore are comparable to each other in terms of

their ability for FIX secretion. Therefore, not only the 100 Fibronectin microcapsules


127

support the osteogenic differentiation of the enclosed MSCs, but also maintain their FIX

secretion capacity. Presence of calcium deposits detected by Alizarin red staining

protocol in microcapsules cultured in osteogenic medium confirms the ability of this

microcapsule system to support the osteogenic differentiation in suspension culture.

However, the encapsulated cells grown in chondrogenic or adipogenic media were not

successfully differentiated into chondorcytes or adipocytes. Our previously designed

fibrinogen-alginate microcapsules were shown to have the same effect on differentiation

of enclosed CB MSCs. Consistently, it was previously reported that cell aggregates are

needed to induce chondrogenic differentiation in MSCs in 3D cultures (A. Goren et al.

2010). The low cell viability in adipogenic media was predicted from our previous

monolayer differentiation studiesfibrinogen paper (submitted paper) and was expected

due to the inefficiency of CB MSCs to differentiate into the adipogenic lineage (Rebelatto

et al. 2008).

5. Conclusion

Fibronectin was incorporated within alginate microcapsules at the concentrations

of 10g/ml (10 Fibronectin), 100g/ml (100 Fibronectin), and 500g/ml (500

Fibronectin) of cell-alginate mixture. Microcapsules were compared in terms of their

effect on cell viability, proliferation as well as FIX secretion from the encapsulated FIX-

engineered CB MSCs. 100 Fibronectin and 500 Fibronectin were shown to have a

significant effect on enhancement of cell viability, proliferation and subsequent FIX


128

secretion. 10 Fibronectin was comparable to non-supplemented microcapsules (control)

and did not significantly affect cell viability or protein secretion.

Differentiation studies confirm that while cultured in appropriate media, the novel

100 Fibronectin alginate microcapsules support osteogenic but not chondrogenic or

adipogenic differentiation of the encapsulated MSCs grown in suspension culture.

Therefore, 100 and 500 fibronectin-alginate matrix encapsulating engineered MSCs have

potentials for cell-based gene therapy of hemophilia B or other protein deficiency

diseases. Future works should address the characterization of cell-matrix interactions

mechanisms behind improved MSC viability and functionality in 100 and 500 Fibronectin

microcapsules cultured in basal or osteogenic media.

Acknowledgements

This work was funded in part by a grant from Canadian Blood Services to G. H. and

A.J.W. Thanks to Marcia Reid and Marnie Timleck for processing the TEM and SEM

samples.

Declaration of Interest



129

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Dolatshahi-Pirouz, A. et al., 2011. Cell shape and spreading of stromal (mesenchymal)

stem cells cultured on fibronectin coated gold and hydroxyapatite surfaces.

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Official Publication of the Federation of American Societies for Experimental

Biology, 24(1), pp.22–31.






pp.1281–1288.







Kilian, K.A. et al., 2010. Geometric cues for directing the differentiation of mesenchymal

stem cells. Proceedings of the National Academy of Sciences of the United States

of America, 107(11), pp.4872–4877.


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York, N.Y.), 294(5550), pp.2268–2269.

McBeath, R. et al., 2004. Cell shape, cytoskeletal tension, and RhoA regulate stem cell

lineage commitment. Developmental Cell, 6(4), pp.483–495.






135(3), pp.203–210.


pp.104–107.



Rebelatto, C.K. et al., 2008. Dissimilar differentiation of mesenchymal stem cells from

bone marrow, umbilical cord blood, and adipose tissue. Experimental Biology and

Medicine (Maywood, N.J.), 233(7), pp.901–913.



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Schoen Fredrick J. & Mitchell Richard N., Tissue, the extracellular matrix, and cell-

biomaterial interactions. In Biomaterials Science: An Introdcution to Materials in

Medicine. Elsevier Academic Press, pp. 260–281.



Son, B.-R. et al., 2006. Migration of bone marrow and cord blood mesenchymal stem

cells in vitro is regulated by stromal-derived factor-1-CXCR4 and hepatocyte

growth factor-c-met axes and involves matrix metalloproteinases. Stem Cells

(Dayton, Ohio), 24(5), pp.1254–1264.


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Stupack, D.G. & Cheresh, D.A., 2002. Get a ligand, get a life: integrins, signaling and

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Whitlock, B.B. et al., 2000. Differential roles for alpha(M)beta(2) integrin clustering or

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CHAPTER 6: SUMMARY AND RECOMMENDATIONS FOR FUTURE WORK

6.1 Summary

Inadequate cell-matrix interactions and subsequent cell death are among the most

critical factors that limit the application of cell microencapsulation techniques in

treatment of hemophilia B or other protein deficiency diseases. In this work, we

hypothesized that decorating the inner core of alginate microcapsules with cell-adhesive

molecules, either proteins or peptides, enhances the viability of the enclosed FIX-

engineered mesenchymal stem cells and may increase the FIX secretion profile. This

hypothesis is supported by extensive studies that have addressed the significance of

selected cell-adhesive proteins and peptides in mediating survival signals (Hersel et al.

2003; Dwayne G Stupack & David A Cheresh 2002; Orive et al. 2009a; Karoubi et al.

2009a). In the work presented in this thesis, biomimetic fibrinogen-, RGD-, or

fibronectin-modified alginate-based microcapsules were designed and constructed to

enhance the viability and functionality of the enclosed FIX-engineered MSCs for future

applications in hemophilia B treatment.

In the first part of this work (Chapter 3), alginate microcapsules were

supplemented with human plasma fibrinogen. After analyzing the cell-loaded

microcapsules using cell viability, proliferation, FIX secretion, and cell-matrix analysis

experiments, it was demonstrated that compared with non-supplemented alginate

microcapsules, bioactive fibrinogen-alginate microcapsules proposed in this work

enhanced cell viability, proliferation and FIX secretion from the enclosed FIX-engineered


133

MSCs. Moreover, our differentiation studies suggested that fibrinogen-alginate

microcapsules provided an appropriate suspension culture for osteogenic, but not

chondrogenic or adipogenic differentiation.

In a subsequent part of this thesis (Chapter 4), engineered MSCs were

encapsulated in RGD-coupled alginate microcapsules. As fibrinogen-alginate, RGD-

alginate microcapsules significantly improved the viability of encapsulated MSC but did

not have a significant effect on the cell proliferation and subsequent FIX secretion from

the encapsulated MSC. Viability and FIX secretion data of encapsulated cells suggested

that although both RGD-alginate and fibrinogen-alginate microcapsules provided

biomimetic 3-D environment for encapsulated MSC applicable in cell therapy

technologies, fibrinogen-alginate microcapsules more significantly enhanced the

proliferation and protein secretion from the encapsulated cells.

In the final part of this research, alginate microcapsules were supplemented with

human plasma fibronectin. Here, we further investigated how the different concentrations

of fibronectin can affect cell viability, proliferation and FIX secretion. Alginate

microcapsules were supplemented with fibronectin at the concentrations of 10g/ml (10

Fibronectin), 100g/ml (100 Fibronectin), and 500g/ml (500 Fibronectin) of cell-

alginate mixture. 100 Fibronectin and 500 Fibronectin were shown to have a significant

effect on enhancement of cell viability, proliferation and FIX secretion. 10 Fibronectin

did not significantly affect cell viability or protein secretion compared to non-

supplemented (control) microcapsules. Differentiation studies confirm that while cultured

in appropriate media, fibronectin-alginate microcapsules support osteogenic but not


134

chondrogenic or adipogenic differentiation of the encapsulated MSCs grown in

suspension culture.

Overall, from the research presented in this thesis, it is concluded that construction

of biomimetic alginate microcapsules using cell-adhesive molecules including

supplemented fibrinogen or fibronectin proteins or cross-linked RGD peptide improve the

viability of the encapsulated FIX-engineered MSCs. In both fibrinogen- and fibronectin-

alginate microcapsules, MSCs demonstrated improved proliferation and FIX secretion

compared to non-supplemented alginate microcapsules. Moreover, our differentiation

studies for fibrinogen- and fibronectin-alginate microcapsules suggested that our protein

modified microcapsules support osteogenic, but not chondrogenic or adipogenic

differentiation of encapsulated MSCs when cultured in appropriate differentiation media.

Even though RGD-alginate microcapsules considerably enhanced the viability of the

enclosed cells, they did not significantly affect the cell proliferation and FIX secretion.

Viability and FIX secretion data of encapsulated cells suggested that although fibrinogen-

alginate, fibronectin-alginate and RGD-alginate microcapsules provided biomimetic 3-D

environment for encapsulated MSC applicable in cell therapy technologies, fibrinogen-

alginate and fibronectin-alginate microcapsules more significantly enhanced the

proliferation and protein secretion from the encapsulated cells and may have potential for

cell-based gene therapy of hemophilia B and other inherited or acquired protein

deficiencies.


135

6.2 Recommendations for Future Work

This work has demonstrated the advantage of using selected cell-adhesive

molecules to create biomimetic microcapsules for enhanced viability and functionality of

the enclosed cells. Various aspects of this study can be investigated in more detail. Also

several avenues for further research can be envisaged.

1. Most of the work presented in this thesis was focused on creating biomimetic

microcapsules for enhanced viability of the enclosed MSCs for cell therapy

applications. Further studies should address the mechanisms behind the

improved MSC viability and functionality. Although MSCs are one of the

most popular candidates for cell therapy applications (S. Wang et al. 2011b),

little is so far known about the phenotype and gene expression of these cells in

three-dimensional environment (Duggal et al. 2009a). Previous studies have

reported that integrins vand likely play a predominant role in the

survival of adherent MSCs (Karoubi et al. 2009a). Integrin expression

profiling of the encapsulated MSCs grown in the modified microcapsules in

this study could illustrate expression of integrins responsible for specific cell

phenotype. Characterization of the biological interactions of MSCs with the

modified matrix can result in a more comprehensive understanding of their

behavior in contact with the extracellular matrix. This can result in detection

of critical integrins that activate intercellular pathways responsible for desired

cell behavior such as cell survival, proliferation, differentiation, migration.

Modification of the extracellular matrix with the ligands specific with these


136

integrins can potentially result in design of biomaterials, which can

specifically induce desired cell behavior.

2. This research thesis focused on the optimization of the microcapsule

environment to achieve high cell viability and FIX secretion in vitro before

starting in vivo experiments. The next step of this study should involve

monitoring the FIX release from the encapsulated cells in vivo and compare

with the secretion value from non-encapsulated cells. Moreover, a therapeutic

demonstration of FIX-MSCs in a mouse model of deficiency would greatly

enhance the significance of this work. This will evaluate a therapeutic

composition of cellular capsules.

6.3 references:



Hersel, U., Dahmen, C. & Kessler, H., 2003. RGD modified polymers: biomaterials for

stimulated cell adhesion and beyond. Biomaterials, 24(24), pp.4385–4415.





135(3), pp.203–210.

Stupack, D.G. & Cheresh, D.A., 2002. Get a ligand, get a life: integrins, signaling and

cell survival. Journal of Cell Science, 115(Pt 19), pp.3729–3738.

Wang, S., Qu, X. & Zhao, R.C., 2011. Mesenchymal stem cells hold promise for

regenerative medicine. Frontiers of Medicine, 5(4), pp.372–378.


137

APPENDIX A : Experimental Methods

The experimental procedures used in this work are explained in detail in this appendix.

A.1 Cell encapsulation procedure

Cells were mixed with alginate solution in a syringe to make a cell-alginate

suspension with the final concentration of 3×106 cells/ml and 1.56% alginate).

Microencapsulation was performed with an encapsulator (Nisco Engineering Inc., Zurich,

Switzerland) as previously described. Cell-alginate mixture was extruded through number

35 needle with a syringe pump (0.9 ml/min). Cell encapsulated alginate beads were

dropped in 1.1% CaCl2 solution. In contact, the droplets gelled. 7kV voltage between the

needle tip and the collecting vial resulted in constructing beads with an average diameter

of 350-450 µm. The outer alginate layer was chemically cross-linked with poly-L-lysine

hydrobromide (PLL; Sigma, St. Louis, MO) for 6 min, and then with another layer of

alginate (Figure A.1).


138

Figure A-1 : Cell encapsulation procedure

A.2 LIVE/DEAD viability kit for encapsulated cells

LIVE/DEAD cell Viability kit (Invitrogen, Burlington, ON, Canada) was used in

chapter 5 of this research to measure the cell viability and proliferation values

A.2.1 Determine the optimal dye concentration

To obtain the best results, the dye concentrations were adjusted to achieve distinct

labeling of live or dead encapsulated MSC with calcein AM and Ethidium homodimer-1

(EthD-1), respectively. Dead cells were prepared by killing the encapsulated cells by


139

treatment with 70% methanol for 30 minutes. Using samples of dead cells, the saturating

concentration of EthD-1 (the lowest concentration that yields maximum fluorescence)

was determined. Using samples of dead cells, concentrations of calcein AN that give

negligible staining of dead cells were determined. Using samples of live cells, the

concentration of calcein AM that gave fluorescence in live cells sufficient to permit clear

detection was determined.

A.2.2 Sample preparation

Viability and proliferation of encapsulated cells using the LIVE/DEAD cell

viability kit was assessed using the manufacturer’s instructions (Invitrogen, Burlington,

ON, Canada) adapted to encapsulated cells. Briefly, samples of microcapsules were

gently washed with tissue-grade PBS and placed in 96-well plate. 100µl of the

LIVE/DEAD reagents were added to the wells to the optimal final concentration of EthD-

1 (5 µM) and calcein AM (2 µM). The samples were incubated at room temperature for

45 minutes. The fluorescence in the experimental and control cell samples was measured

using the appropriate excitation and emission filters:

A. Fluorescence at 645 nm in the experimental cell sample, labeled with calcein AM

and EthD-1 = F(645)sam

B. Fluorescence at 530 nm in the experimental cell sample, labeled with calcein AM

and EthD-1 = F(530)sam

C. Fluorescence at 645 nm in a sample where all cell are dead, labeled with EthD-1

only = F(645)max


140

D. Fluorescence at 645 nm in a sample where all cells are dead, labeled with calcein

AM only = F(645)min

E. Fluorescence at 530 nm in a sample where all (or nearly all) cell are alive, labeled

with EthD-1 only = F(530)min

F. Fluorescence at 530 nm in a sample where all (or nearly all) cell are alive, labeled

with calcein AM only = F(530)max

G. Fluorescence at 530 nm of the cell-free sample with or without dye added =

F(530)0

H. Fluorescence at 645 nm of the cell-free sample with or without dye added =

F(645)0

A.2.3 Determining absolute numbers of live and dead cells

The total number of cells in a sample was counted by killing all of the cells as mentioned

above, labeling with a saturating concentration of EthD-1 and measuring fluorescence at

>600 nm. The intensity was then linearly related to the total number of cells present in

the sample.

A.2.4 Interpretation of the results:

As defined in the protocol (Invitrogen, Burlington, ON, Canada), the percentage of dead

cells were calculated from the florescence readings defined above as:

% Dead Cells = min

max min

(645) (645)100%

(645) (645)

samF F

F F

%Live Cells=100% - %Dead Cells


141

A.3 Sandwich-style FIX ELISA

Antibody to FIX was coated onto the wells of a microtitre plate. The plates were

washed and the media samples containing FIX were applied. The coated antibody

captured the FIX present in the sample. The plate was then washed to remove the

unbound material and a peroxidase conjgated secondary antibody to FIX was added to

the wells to bind to the captured antibody. The plate was once again washed to remove

the unbound conjugated antibody and incubated with o-phenylenediamine (OPD) to

express the peroxide activity. The reaction was then quenched with addition of 2.5 M

H2SO4 and the produced color was quantified using a microplate reader. The color

generated was proportional to the level of FIX present in the sample (Affinity Biologicals

Inc. FIX ELISA protocol)

Coating Buffer (50 mM carbonate):

1.59g Na2CO3 and 2.93g NaHCO3 up tp 1 litre. pH adjusted to 9.6

Wash Buffer (PBS-Tween 0.1%, v/v):

1.0 ml of Tween-20 to 1 litre pf PBS. pH adjusted to 7.4

Sample Diluent (HBS-BSA-EDTA-T20):

5.95g HEPES (free acid), 1.46g NaCl, 0.93g Na2EDTA, 2.5g Bovine Serum

Albuin (Sigma, RIA grade) dissolved in 200 ml H2O. 0.25 ml of tween-20 was

added and pH was adjusted to 7.2 with NaOH nd made uo to a final volume of

250 ml with H2O

Substrate Buffer (Citrate-Pjosphate buffer pH 5.0):

2.6g citric acid and 6.9g Na2HPO4 up to a final volume of 500 ml with H2O

OPD Substrate (o-Phenylenediamine.2HCl):


142

5mg tablets (Sigma) dissolved in 12 ml substrate buffer. 12l 30% H2O2 was

added.

Stopping Solution (2.5 M H2SO4):

13.9 ml of 18 Molar sulphuric acid stock solution to 86 ml H2O

A.3 Statistical analysis

Data are expressed as means ± SD. When an average value (A) was normalized in

respect to another average value (B), the standard deviation of the reported value (X=A/B)

was calculated as:

2 2dX dA dB

X A B

Where dX, dA, and dB are standard deviation of values X, A, and B, respectively.


differences existed in the groups of data. Where several mean values were tested pair-

wise, Turkey HSD was conducted and when only two mean values were compared,

Student’s t-test was conducted as post hoc tests to compare the pairs of data. Differences

were considered significant when p<0.05.


143

APPENDIX B : Publications and Awards

B.1 Journal Articles

Sayyar B, Dodd M, Marquez-Curtis L., Janowska-Wieczorek A., Hortelano

G.,”Encapsulation of FIX-Engineered Human Mesenchymal Stem Cells in RGD-Alginate

vs. Fibrinogen-Alginate Microcapsules for Enhanced FIX Secretion”, (To be Submitted),

2012

Sayyar B, Dodd M., Marquez-Curtis L., Janowska-Wieczorek A., Hortelano G,

“Fibronectin-Alginate Microcapsules Improve Cell Viability and Protein Secretion of

Encapsulated FIX-Engineered Human Mesenchymal Stem Cells”, (To be Submitted),

2012

Sayyar B, Dodd M., Wen J, Ma S, Marquez-Curtis L., Janowska-Wieczorek A.,

Hortelano G, “Encapsulation of Factor IX Engineered Mesenchymal Stem Cells in

Fibrinogen-Alginate Microcapsules Enhances their Viability and Transgene Secretion”,

Journal of Tissue Engineering (Accepted), 2012

Sun J, Sayyar B, Butler JE, Pharkya P, Fahland TR, Famili I, Schilling CH, Lovley DR,

Mahadevan R., “Genome-scale constraint-based modeling of Geobacter

metallireducens”, BMC Syst Biol. 2009 Jan 28;3:15

B.2 Refereed Conference Presentations

Sayyar B., Dodd M., Marquez-Curtis L., Janowska-Wieczorek A., Hortelano G,

Fibronectin-alginate microcapsules improve cell viability and protein secretion of

encapsulated FIX-engineered human MSC, XX International Conference on

Bioencapsulation, Orillia, Ontario, Canada, 2012

Sayyar B, Dodd M., Marquez-Curtis L., Janowska-Wieczorek A., Hortelano

GFibrinogen and RGD-coupled Alginate Microcapsules for Encapsulation of

Mesenchymal Stem Cells, 9th

World Biomaterial Congress, Chengdu, China, 2012

Sayyar B, Dodd M, Wen J, Ma S, Marquez-Curtis L, Janowska A, Hortelano G,

Fibrinogen-coupled Alginate Microcapsules for Enhanced Survival and Functionality of

Mesenchymal Stem Cells, 29th Canadian Biomaterials Society Meeting, Vancouver,

2011

Sayyar B, Mahadevan R. Electron Equivalent Models to Accommodate. Geobacter

Species, American Society for Microbiology (ASM), Boston, 2008

http://www.ncbi.nlm.nih.gov.libaccess.lib.mcmaster.ca/pubmed?term=Sun%20J%5BAuthor%5D&cauthor=true&cauthor_uid=19175927

http://www.ncbi.nlm.nih.gov.libaccess.lib.mcmaster.ca/pubmed?term=Sayyar%20B%5BAuthor%5D&cauthor=true&cauthor_uid=19175927

http://www.ncbi.nlm.nih.gov.libaccess.lib.mcmaster.ca/pubmed?term=Butler%20JE%5BAuthor%5D&cauthor=true&cauthor_uid=19175927

http://www.ncbi.nlm.nih.gov.libaccess.lib.mcmaster.ca/pubmed?term=Pharkya%20P%5BAuthor%5D&cauthor=true&cauthor_uid=19175927

http://www.ncbi.nlm.nih.gov.libaccess.lib.mcmaster.ca/pubmed?term=Fahland%20TR%5BAuthor%5D&cauthor=true&cauthor_uid=19175927

http://www.ncbi.nlm.nih.gov.libaccess.lib.mcmaster.ca/pubmed?term=Famili%20I%5BAuthor%5D&cauthor=true&cauthor_uid=19175927

http://www.ncbi.nlm.nih.gov.libaccess.lib.mcmaster.ca/pubmed?term=Schilling%20CH%5BAuthor%5D&cauthor=true&cauthor_uid=19175927

http://www.ncbi.nlm.nih.gov.libaccess.lib.mcmaster.ca/pubmed?term=Lovley%20DR%5BAuthor%5D&cauthor=true&cauthor_uid=19175927

http://www.ncbi.nlm.nih.gov.libaccess.lib.mcmaster.ca/pubmed?term=Mahadevan%20R%5BAuthor%5D&cauthor=true&cauthor_uid=19175927

http://www.ncbi.nlm.nih.gov.libaccess.lib.mcmaster.ca/pubmed?term=bahareh%20sayyar


144

B.3 Other Conference Presentations

Sayyar B, Hortelano G, Biomimetic alginate-based microcapsules for hemophilia

treatment, BME Symposium, Hamilton, 2012

Sayyar B, Hortelano G. Cell encapsulation for hemophilia treatment, Poster Presentation,

BME Symposium, Hamilton, 2009

Sayyar B, Mahadevan R. Thermodynamic electron equivalent models for Geobacter

species, Poster presentation, CSChE 9th Annual Ontario Quebec Biotechnology Meeting,

Toronto, 2007

B.4 AWARDS AND SCHOLARSHIPS

Graduate Student Association Travel Grant (500$) 2012 McMaster University

Yates Scholarship Fund (500$) 2012

McMaster University

Canadian Society for Biomaterials-Travel Award (1000$) 2012

Best poster presenter-BME Symposium (100$) 2012

McMaster University

Canadian Society for Biomaterials-Highlighted Researcher of the Month 2011

Canadian Society for Biomaterials-Travel Award 2011

Ontario Graduate Scholarship (OGS) 2011-2012

Queen Elizabeth II Science and Technology Scholarship (OGSST) 2010-2011

International Excellence Award 2009-2010

McMaster University

Graduate Research Scholarship (19500$/year) 2006-2008

University of Toronto

encapsulation of factor ix-engineered ......the work presented in this thesis was focused on design...

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